CN110452962A - It is detected using the target nucleic acid sequence of different detection temperature - Google Patents
It is detected using the target nucleic acid sequence of different detection temperature Download PDFInfo
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- CN110452962A CN110452962A CN201910807436.4A CN201910807436A CN110452962A CN 110452962 A CN110452962 A CN 110452962A CN 201910807436 A CN201910807436 A CN 201910807436A CN 110452962 A CN110452962 A CN 110452962A
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Abstract
It the present invention relates to the use of the target nucleic acid sequence detection of different detection temperature.In the present invention using different detection temperature, multiple target nucleic acid sequences only can detect also with existing embodiment with the label of single type in 1 reaction vessel.In the prior art, multiple target nucleic acid sequences are detected by melting analysis after target amplification.Unlike this, the present invention is not necessarily to carry out melting analysis after target amplification, to significantly shorten analysis time.
Description
The application be the applying date be on December 9th, 2014, it is entitled " using it is different detection temperature target nucleic acid sequences
The divisional application of the Chinese invention patent application No.201480077317.3 of detection ".
Technical field
It the present invention relates to the use of the target nucleic acid sequence detection of different detection temperature.
Background technique
In order to detect target nucleic acid sequence, extensively using monitoring target expands on one side in real time fashion, target nucleic acid can detect on one side
The real-time detection method of sequence.In general, using marked with target nucleic acid sequence specific hybrid in real-time detection method
Probe or primer.As the example of the method using the hybridization between marked probe and target nucleic acid sequence, including the use of having
Molecular beacon (Molecular beacon) method (Tyagi etc., Nature of the probe of the double labeling of hairpin structure
Biotechnology v.14 MARCH 1996), HyBeacon method (French DJ etc., Mol.Cell Probes, 15
(6): 363-374 (2001), be marked as respectively using 2 donor (donor) and receptor (acceptor) 2 probes hybridization
Detecting probe method (Bernad etc., 147-148 Clin Chem 2000；46) and using single labelled oligonucleotides Lux method
(U.S. Patent No. 7537886).In the art extensively using TaqMan method (U.S. Patent No. 5210015 and
No. 5538848), above-mentioned TaqMan method (U.S. Patent No. No. 5210015 and No. 5538848) is using by double labeling
Probe and DNA (DNA) polymerase 5 '-nucleases above-mentioned probe cutting.
The example of method as the primer using label, including sunrise (Sunrise) primed method (Nazarenko etc.,
2516-2521 Nucleic Acids Research, 1997, v.25no.12 and U.S. Patent No. 6117635) and scorpion shape
(Scorpion) primed method (Whitcombe etc., 804-807, v.17 1999 AUGUST Nature Biotechnology
And U.S. Patent No. 6326145) and TSG primed method (WO 2011-078441).
As replacing close to method, propose to utilize by depend on target nucleic acid sequence it is existing in the way of the reality of dimer that is formed
When detection method, for example, invader (Invader) analysis (U.S. Patent No. 5691142, U.S. Patent No. 6358691 and
U.S. Patent No. 6194149), detection and labeled oligonucleotide cutting and extend (PTO cleavage and extension；
PTOCE) method (WO 2012/096523), detection and labeled oligonucleotide cutting and extension-dependent signals transduction few nucleosides
Acid hybridization (PTO Cleavage and Extension-Dependent Signaling Oligonucleotide
Hybridization；PCE-SH) method (WO 2013/115442) and detection and labeled oligonucleotide cutting and extension-dependence
Non- hybridization (the PTO Cleavage and Extension-Dependent Non-Hybridization of property；PCE-NH) method
(PCT/KR2013/012312)。
Existing real-time detection technology as described above is during expanding mutually related or unrelated amplification of signal to target, 1
The signal generated from fluorescent marker is detected at a temperature of a selected detection.According to existing real-time detection technology, reacted at one
It when detecting multiple target nucleic acid sequences, is not distinguished mutually using the label of single type in pipe for the multiple of target nucleic acid sequence generation
Signal.Therefore, in order to detect multiple target nucleic acid sequences, different types of label is generally utilized in existing real-time detection technology.
Using the label of single type, it can detect multiple target nucleic acid sequences using the melting analysis of Tm difference, still, solution
Link analysis has the drawback that the progress time of melting analysis is longer than real-time technique and carries out the time, and with target nucleic acid sequence
Increase, the design of the probe for the Tm value for having different is made to become to be increasingly difficult to.
Therefore, if exploitation utilizes the label of single type and single independent of melting analysis and in 1 reaction vessel
The detector of type, then can be with convenience, cost effectiveness (CE) and efficiency to detect the new method of target nucleic acid sequence or close to method
The mode greatly improved detects multiple target nucleic acid sequences.If also, in conjunction with said new method and other detection methods (for example, solution
Link analysis), then multiple targets can be detected using the label of single type in 1 reaction vessel in such a way that efficiency greatly improves
Nucleic acid sequence.
In this specification, numerous patents and document are had references to, and illustrates its reference with bracket.In order to brighter
Really illustrate that the level of the present invention and the technical field of the invention, the disclosure of this patent and document are fully inserted into this theory
Bright book is as reference.
Summary of the invention
The present inventor in order to develop in 1 reaction vessel using the detector of the label of single type and single type come
It detects the new method of multiple target nucleic acid sequences and has carried out making great efforts research.Result of study, detection temperature of the present inventor in adjusting
After lower acquisition is for the signal of target nucleic acid sequence, being confirmed by suitably analysis detection result can be imitated with convenience, expense
The mode that fruit and efficiency greatly improve is examined in 1 reaction vessel using the detector of the label of single type and single type
Survey multiple target nucleic acid sequences.
Therefore, the object of the present invention is to provide the sides using 2 target nucleic acid sequences in different detection temperature detection samples
Method and kit.
It is a further object of the present invention to provide more come the nucleotide to sample nucleic acid sequence using different detection temperature
The method and kit that state property (SNP) genotype is analyzed.
A further purpose of the present invention is to provide and utilize at least three target nucleic acid sequence in different detection temperature detection samples
Method and kit.
Another object of the present invention is to provide utilize at least three in different detection temperature and melting analysis detection sample
The method and kit of target nucleic acid sequence.
It is a further object of the present invention to provide detect at least three target nucleic acid using detection temperature analysis and melting analysis
The method and kit of sequence.
It is a further object of the present invention to provide computer readable storage medium, comprising the instruction for running processor,
Above-mentioned processor is used to determine the existing method of 2 target nucleic acid sequences in sample using different detection temperature.
It is a further object of the present invention to provide the devices using target nucleic acid sequence in different detection temperature detection samples.
It is a further object of the present invention to provide computer program, run for determining in sample 2 target nucleic acid sequences
The processor of existing method and it is stored in computer readable storage medium.
It is a further object of the present invention to provide computer readable storage medium, comprising the instruction for running processor,
Above-mentioned processor is used to determine the existing method of 3 target nucleic acid sequences in sample using different detection temperature.
Together with range and attached drawing is claimed with appended invention, other mesh of the invention can be made by following detailed description
And advantage definitely.
Detailed description of the invention
Fig. 1 a is to show to detect the target nucleic acid sequence (chlamydia trachomatis with relatively high detection temperature (72 DEG C)
The genomic deoxyribonucleic acid of (CT, Chlamydia trachomatis)), with relatively low detection temperature (60 DEG C)
Target nucleic acid sequence (gonococcus (NG, Neisseria gonorrhoeae) genomic deoxyribonucleic acid) and their combination and
Utilize the figure of the testing result of the invention of different detection temperature.By detection and labeled oligonucleotide cutting and extend poly- in real time
Polymerase chain reacts (PCR) method and generates the signal for being directed to above-mentioned chlamydia trachomatis and gonococcus.
Fig. 1 b is between the signal at a temperature of the signal shown by a temperature of relatively high detection and relatively low detection
Ratio determine have it is relatively low detection temperature target nucleic acid sequence existing figure.
Fig. 1 c be show by the signal at a temperature of relatively high detection and the signal at a temperature of relatively low detection it
Between ratio float determine with it is relatively low detection temperature target nucleic acid sequence existing figure.
Fig. 1 d and Fig. 1 e are the letter at a temperature of the signal shown by a temperature of relatively high detection and relatively low detection
Difference between number determines the existing figure with the target nucleic acid sequence of relatively low detection temperature, by above-mentioned relatively high detection
At a temperature of signal skew at threshold value and for obtaining above-mentioned difference.
Fig. 2 a be show in order to detect with it is relatively high detection temperature (72 DEG C) target nucleic acid sequence (chlamydia trachomatis
Genomic deoxyribonucleic acid), with it is relatively low detection temperature (60 DEG C) target nucleic acid sequence (gonococcus genome deoxidation core
Ribosomal ribonucleic acid) and their combination and utilize it is different detection temperature testing results of the invention figure.Gathered in real time by TaqMan
Polymerase chain reaction method generates the signal for being directed to above-mentioned chlamydia trachomatis, cuts and extends by detection and labeled oligonucleotide
Real-time polymerase chain reaction method generates the signal for being directed to above-mentioned gonococcus.
Fig. 2 b is between the signal at a temperature of the signal shown by a temperature of relatively high detection and relatively low detection
Ratio determine have it is relatively low detection temperature target nucleic acid sequence existing figure.
Fig. 2 c be show by the signal at a temperature of relatively high detection and the signal at a temperature of relatively low detection it
Between ratio float determine with it is relatively low detection temperature target nucleic acid sequence existing figure.
Fig. 2 d and Fig. 2 e are the letter at a temperature of the signal shown by a temperature of relatively high detection and relatively low detection
Difference between number determines the existing figure with the target nucleic acid sequence of relatively low detection temperature, by above-mentioned relatively high detection
At a temperature of signal skew at threshold value and for obtaining above-mentioned difference.
Fig. 3 is to show to have according to using the real-time polymerase chain reaction and being directed to for melting analysis of different detection temperature
The target nucleic acid sequence (genomic deoxyribonucleic acid of chlamydia trachomatis) of relatively high detection temperature (72 DEG C) has relatively
The target nucleic acid sequence (gonococcus genomic deoxyribonucleic acid) of lower detection temperature (60 DEG C) and the detection knot of their combination
The figure of fruit is generated the signal for being directed to above-mentioned chlamydia trachomatis by TaqMan real-time polymerase chain reaction method, passes through detection
It is cut with labeled oligonucleotide and extends real-time polymerase chain reaction method and generate the signal for being directed to above-mentioned gonococcus.
Fig. 4 a is to show to utilize the nucleotide polymorphisms genotyping of the invention of different detection temperature in real time fashion
As a result figure.Methylenetetrahydrofolate reductase (MTHFR) (C677T) human genome deoxidation is used as template (target sequence)
Ribonucleic acid.And detect wild-type homozygote (CC), mutant homozygote (TT) and heterozygote (CT).By detecting and marking
Remember oligonucleotides cutting and extend real-time polymerase chain reaction method and generates all signals.
Fig. 4 b is to show using between the signal at a temperature of relatively high detection and the signal at a temperature of relatively low detection
Ratio nucleotide polymorphisms genotyping figure.
Fig. 5 a to Fig. 5 c is to show to detect the genome of 3 target sequences (genomic deoxyribonucleic acid of gonococcus)
DNA, the genomic deoxyribonucleic acid of chlamydia trachomatis and mycoplasma genitalium (MG, Mycoplasma
Genitalium genomic deoxyribonucleic acid)) and the figure of the testing results of the invention using different detection temperature.Pass through
Detection and labeled oligonucleotide cutting and extension real-time polymerase chain reaction method generate and are directed to above-mentioned chlamydia trachomatis and leaching
The signal of coccus.Consider signal generating mechanism, " 95 DEG C " is selected as the signal detection temperature to mycoplasma genitalium, as to sand
The signal detection temperature of chlamydia oculogenitale selects " 72 DEG C ", selects " 60 DEG C " as the signal detection temperature to gonococcus
Fig. 5 d is to show in order to determine the presence of chlamydia trachomatis genomic deoxyribonucleic acid and at 95 DEG C and 72 DEG C
At a temperature of the figure of End- △ RFU that is calculated using Relative fluorescence units (RFU) value of endpoint.
Fig. 5 e is to show in order to determine the presence of gonococcus genomic deoxyribonucleic acid and in 72 DEG C and 60 DEG C of temperature
The lower RFU value using endpoint is come the figure of the End- △ RFU calculated.
Specific embodiment
Maximum feature of the invention is that the detection of the label and single type of single type is utilized in 1 reaction vessel
Device detects multiple target nucleic acid sequences.In the present invention using different detection temperature, with single type in 1 reaction vessel
Also multiple target nucleic acid sequences be can detect in the case where label.Feature selecting according to the present invention structure of the invention, and become and use
In the surprising technique of detection target nucleic acid sequence.
In existing real-time polymerase chain reaction method, 2 seed types are needed in order to detect 2 target nucleic acid sequences
Fluorescent marker or melting analysis.
Present invention can be implemented in 2 target nucleic acid sequences are detected using the fluorescent marker of single type in 1 reaction vessel
Real-time polymerase chain reaction scheme.With selecting a property, the present invention detects 2 target nucleic acid sequences using real-time polymerase chain reaction
A sequence in column, and another sequence in 2 target nucleic acid sequences is detected using melting analysis, thus 2 detectable
Target nucleic acid sequence.
The present invention using can according to for target nucleic acid sequence signal-generation mechanism, by temperature adjustment signal detection
The result of study of the present inventor.
For example, when the signal-in order to detect the first target nucleic acid sequence and hybridization of the use based on target nucleic acid sequence and probe
When generation mechanism, can produce at a temperature of probe hybridizes with the first target nucleic acid sequence indicates letter existing for the first target nucleic acid sequence
Number, to can detect that.In contrast to this, probe and the first target nucleic acid sequence without hybridize at a temperature of do not generate letter
Number, to can not detect.It is found that there is the temperature for generating signal according to signal-generation mechanism and can not in this viewpoint
Generate the temperature of signal.
In this signal-generation mechanism, the temperature that can hybridize probe and the first target nucleic acid sequence is as first
The detection temperature of target nucleic acid sequence uses.And it can not be using the non-hybridized temperature of probe and the first target nucleic acid sequence as detection temperature
Degree uses.
It, can be according to the temperature that there is generation signal when using hybridization probe also, in order to detect the second target nucleic acid sequence
The fact that range and the temperature range that can not generate signal, determines the detection temperature of the second target nucleic acid sequence.
When the Tm value of the probe used to detect the second target nucleic acid sequence is less than for detecting the first target nucleic acid sequence
Probe Tm value when, can not generate for the second target nucleic acid sequence signal it is relatively higher at a temperature of detection for the
The signal of one target nucleic acid sequence.That is, signal is generated between 2 signal-generation mechanisms for 2 target nucleic acid sequences, and
It is had differences in detected temperature.
When above-mentioned 2 target nucleic acid sequences coexist in sample, exist generate for the first target nucleic acid sequence signal and
The temperature range of the signal for the second target nucleic acid sequence can not be generated.On the other hand, in the temperature for being lower than above-mentioned temperature range
The signal for being directed to above-mentioned 2 target nucleic acid sequences is generated under range.
The detection temperature for being directed to each target nucleic acid sequence can be determined according to 2 temperature ranges.It can be from the former temperature range
Relatively high detection temperature is selected, above-mentioned relatively high detection temperature is distributed in the first target nucleic acid sequence.It can be from the temperature of the latter
It spends range and selects relatively low detection temperature, above-mentioned relatively low detection temperature is distributed in the second target nucleic acid.
According to the present invention, the letter at a temperature of relatively high detection is measured in order to determine the presence of the first target nucleic acid sequence
Number.According to the present invention, the detection at a temperature of above-mentioned relatively high detection can determine the presence of the first target nucleic acid sequence.
Important technical characteristic of the invention is as follows: in order to determine the second target nucleic acid sequence with relatively low detection temperature
The presence of column and confirmed using the signal at a temperature of relatively high detection and the signal at a temperature of relatively low detection in phase
To the signal detected at a temperature of lower detection.
With selecting a property, the present inventor can be by detecting the first target using different detection temperature at a temperature of relatively high detection
Nucleic acid sequence, and can be by detecting the second target nucleic acid sequence using the melting analysis close to method is generated as another signal.
During real-time detection process, the signal-generation mechanism used for melting analysis can provide signal at a certain temperature.Cause
This, even if in the case where detecting a sequence in 2 target nucleic acid sequences by melting analysis, 2 target nucleic acid sequences
It needs that there is mutually different detection temperature.
The present invention can be achieved as following numerous embodiments:
(a) detection of 2 target nucleic acid sequences in the sample of different detection temperature is utilized；
(b) the nucleotide polymorphisms genotyping of the sample nucleic acid sequence of different detection temperature is utilized；
(c) detection of at least three target nucleic acid sequence in the sample of different detection temperature is utilized；
(d) detection of 2 target nucleic acid sequences in the sample of different detection temperature and melting analysis is utilized；And
(e) detection of at least three target nucleic acid sequence in the sample of detection temperature analysis and melting analysis is utilized.
I. the detection of 2 target nucleic acid sequences in the sample of different detection temperature is utilized
In one embodiment of the present invention, the present invention, which provides, utilizes 2 target nucleic acids in different detection temperature detection samples
The method of sequence, comprising: step (a), signal-generation in 1 reaction vessel and for detecting above-mentioned 2 target nucleic acid sequences
Said sample is cultivated together by mechanism (signal-generating means), and is produced using the detection of the detector of single type
Raw signal detects each above-mentioned target nucleic acid sequence by corresponding signal-generation mechanism, in above-mentioned 2 target nucleic acid sequences
One has relatively high detection temperature (the relatively high determined by corresponding above-mentioned signal-generation mechanism
Detection temperature), another has the relatively low inspection determined by corresponding above-mentioned signal-generation mechanism
Testing temperature (relatively low detection temperature), above-mentioned relatively high detection temperature are that can generate needle
To the temperature of the signal of the target nucleic acid sequence with above-mentioned relatively high detection temperature, above-mentioned relatively low detection temperature is can
It generates for the signal of the target nucleic acid sequence with above-mentioned relatively low detection temperature and for above-mentioned relatively the Supreme People's Procuratorate
The temperature of the signal of the target nucleic acid sequence of testing temperature, the signal generated by above-mentioned 2 signals-generation mechanism is not by above-mentioned unitary class
The detector of type is distinguished, and carries out above-mentioned inspection at a temperature of above-mentioned relatively high detection temperature and above-mentioned relatively low detection
It surveys；And step (b), the presence of 2 target nucleic acid sequences is determined according to the signal detected in above-mentioned steps (a)；(i) basis
The signal detected at a temperature of above-mentioned relatively high detection determines the target nucleic acid sequence with above-mentioned relatively high detection temperature
Presence, (ii) according to it is above-mentioned it is relatively high detection temperature detection go out signal and at a temperature of above-mentioned relatively low detection
Difference between the signal detected determines the presence with the target nucleic acid sequence of above-mentioned relatively low detection temperature.
According to the existing real-time polymerase chain reaction method using amplification curve, it is not distinguished using offer identical
Signal-generation mechanism of signal distinguishes multiple target nucleic acid sequences, to can not be detected, this is the neck of technology belonging to the present invention
The common sense in domain.
The present invention overcomes limitation relevant to the common sense of the art, provides and detects target in a manner of greatly improving
The result that do not expect of nucleic acid sequence.
Steps are as follows that the present invention will be described in detail by each.
Step (a): with the culture of signal-generation mechanism and detection signal
Firstly, being trained together with signal-generation mechanism for detecting above-mentioned 2 target nucleic acid sequences in 1 reaction vessel
After the feeding sample to be analyzed, generated signal is detected using the detector of single type.By above-mentioned 2 signals-generation machine
The signal that structure generates is not distinguished by the detector of above-mentioned single type.
The present invention utilizes signal-generation mechanism to generate the signal for target nucleic acid sequence.By corresponding signal-
Generation mechanism detects each target nucleic acid sequence.Term " signal-generation mechanism (signal- used in the present specification
Generating means) " mean used during generating for indicating signal existing for target nucleic acid sequence appoint
What substance (any material), such as comprising oligonucleotides, label and enzyme.With selecting a property, it is used in the present specification
Term " signal-generation mechanism " mean to utilize any method (any method) of the substance for generating signal.
An example according to the present invention, culture be under conditions of can produce signal by above-mentioned signal-generation mechanism into
Capable.This condition includes the temperature, salinity and pH of solution.
The example of the oligonucleotides used as signal-generation mechanism includes the few nucleosides with target nucleic acid sequence specific hybrid
Sour (for example, probe and primer), when the probe or primer that hybridize with target nucleic acid sequence release segment by cutting, as above-mentioned
The oligonucleotides that signal-generation mechanism uses includes the capture oligonucleotides (capture with above-mentioned segment specific hybrid
Oligonucleotide), when extending with the segment of above-mentioned capture oligonucleotide hybridization and form extended chain, as above-mentioned letter
Number-oligonucleotides that uses of generation mechanism includes oligonucleotides with above-mentioned extended chain specific hybrid, as above-mentioned signal-production
The oligonucleotides that life structure uses includes the oligonucleotides with above-mentioned capture oligonucleotides specific hybrid, as above-mentioned signal-
The oligonucleotides that generation mechanism uses includes their combination.
Even if signal production principle is identical, the signal generating mechanism comprising not homotactic oligonucleotides can also be considered as mutually
It is different.
Above-mentioned label can be connected with oligonucleotides or exist with free form (free form).In the extension phase
Between above-mentioned label be inserted into extension products (extended products).
When using the cutting of above-mentioned oligonucleotides during signal generates, the example as above-mentioned enzyme includes 5 '-nucleic acid
Enzyme, 3 '-nucleases especially include that nucleic acid polymerase with 5 '-nucleases, the nucleic acid with 3 '-nucleases are poly-
Synthase or FEN nuclease.
In the present invention, signal is generated in many ways using above-mentioned many kinds of substance.
It is to be produced in a manner of being formed dependent on dimer at least one in 2 signal-generation mechanisms according to an example
Signal-generation mechanism of raw signal.
According to an example, signal-generation mechanism for each above-mentioned target nucleic acid sequence is to be formed dependent on dimer
Mode generate signal-generation mechanism of signal.
According to an example, above-mentioned dimer includes double chain target acid sequence.
It is right in this explanation, the expression being used together with signal-generation mechanism " is produced in a manner of being formed dependent on dimer
Raw signal " means in a manner of dependent on the combination (association) of 2 nucleic acid molecules or dissociation (dissociation)
The signal being detected is provided.Above-mentioned expression includes the dimer formed in a manner of existing for dependent on target nucleic acid sequence
(for example, detection oligonucleotides and nucleic acid sequence comprising label) provides signal.Also, above-mentioned expression includes by inhibiting dimerization
The hybridization of object (for example, the markd detection oligonucleotides of tool and nucleic acid sequence) provides signal, above-mentioned inhibition with other two
The formation of polymers generates.
In particular, passing through two between target nucleic acid sequence and the detection oligonucleotides of above-mentioned target nucleic acid sequence specific hybrid
The formation of polymers generates above-mentioned signal.
Term " detection oligonucleotides (detection oligonucleotide) " used in the present specification is ginseng
With the oligonucleotides for generating detected signal.An example according to the present invention, above-mentioned detection oligonucleotides include to participate in generating
The oligonucleotides of substantial signal.For example, other oligonucleotides are (for example, comprising complementary with target nucleic acid sequence or detection oligonucleotides
Nucleotide sequence oligonucleotides) and detection oligonucleotides hybridization or non-hybridization determine signal generation.
An example according to the present invention, above-mentioned detection oligonucleotides include at least 1 label.
The signal formed based on the dimer between target nucleic acid sequence and detection oligonucleotides can be by including scorpion shape method
(Whitcombe etc., Nature Biotechnology 17:804-807 (1999)), sunrise (or Amplifluor) method
(Nazarenko etc., Nucleic Acids Research, 25 (12): 2516-2521 (1997) and U.S. Patent No. 6117635
Number), LUX method (U.S. Patent No. 7537886), plessor (Plexor) method (Sherrill CB etc., Journal of
The American Chemical Society, 126:4550-4556 (2004)), method of molecular beacons (Tyagi etc.,
Nature Biotechnology v.14MARCH 1996), Hybeacon method (French DJ etc., Mol.Cell
Probes, 15 (6): 363-374 (2001)), adjacent hybridization probe method (Bernard P.S. etc., Anal.Biochem.,
273:221 (1999)) and LNA method (U.S. Patent No. 6977295) a variety of methods generate.
In particular, by depend on the mediation oligonucleotides (mediation with above-mentioned target nucleic acid sequence specific hybrid
Oligonucleotide the dimer that the mode of cutting) is formed generates above-mentioned signal.
Term " mediating oligonucleotides " used in the present specification is to mediate to be formed not including the two of target nucleic acid sequence
The oligonucleotides of polymers.
The cutting itself of an example according to the present invention, above-mentioned mediation oligonucleotides does not generate signal, few mediate
After the hybridization and cutting of nucleotide, the segment formed by above-mentioned cutting participates in the successive reaction for generating signal.
According to an example, the hybridization or cutting of above-mentioned mediation oligonucleotides itself do not generate signal.
An example according to the present invention, above-mentioned mediation oligonucleotides are put in a manner of hybridizing with target nucleic acid sequence or being cut
Segment out, to include the oligonucleotides for mediating dimer to generate.It is based in particular, above-mentioned segment mediates in capturing oligonucleotides
The formation of the dimer of the extension of above-mentioned segment.
An example according to the present invention, above-mentioned mediation oligonucleotides includes: (i) 3 '-targeting moiety, includes and target nucleic acid sequence
Arrange complementary hybridizing nucleotide sequence；And (ii) 5 '-mark position, comprising with the non-complementary nucleotides sequence of target nucleic acid sequence
Column.
Segment, above-mentioned segment and the few core of capture are released in an example according to the present invention, the cutting of above-mentioned mediation oligonucleotides
Thuja acid specific hybrid, and extend in above-mentioned capture oligonucleotides.
An example according to the present invention, the mediation oligonucleotides hybridized with target nucleic acid sequence release segment by cutting, on
It states segment and captures oligonucleotides specific hybrid, above-mentioned segment forms extended chain in a manner of extending, this results in above-mentioned
Extension dimer between extended chain and capture oligonucleotides, so that providing indicates signal existing for target nucleic acid sequence.
An example according to the present invention, few using the third comprising the hybridizing nucleotide sequence complementary with above-mentioned extended chain
In the case where nucleotide, the hybridization of above-mentioned third oligonucleotides and extended chain is by forming other kinds of dimer, to provide
Indicate signal existing for target nucleic acid sequence.
An example according to the present invention includes the hybridizing nucleotide sequence complementary with above-mentioned capture oligonucleotides using
In the case where third oligonucleotides, the dimer between above-mentioned third oligonucleotides and above-mentioned capture oligonucleotides is formed by upper
The inhibition that the dimer between extended chain and above-mentioned capture oligonucleotides is formed is stated, so that providing indicates existing for target nucleic acid sequence
Signal.
An example according to the present invention, above-mentioned segment, above-mentioned extended chain, above-mentioned capture oligonucleotides, above-mentioned third widow core
Thuja acid or their combination can play the role of detecting oligonucleotides.
Signal based on the dimer formed in a manner of the cutting dependent on above-mentioned mediation oligonucleotides can be by including
Detection and labeled oligonucleotide cutting and extend (PTO cleavage and extension) method (WO 2012/096523),
Detection and labeled oligonucleotide cutting and extension-dependent signals transduction oligonucleotide hybridization (PTO Cleavage and
Extension-Dependent Signaling Oligonucleotide Hybridization) method (WO 2013/
115442) and detection and labeled oligonucleotide cutting and non-hybridization (the PTO Cleavage and of extension-dependence
Extension-Dependent Non-Hybridization) method (PCT/KR2013/012312) a variety of methods generate.
Relatively with the term disclosed in above-mentioned bibliography, it is corresponding with oligonucleotides for example under: mediate few
Nucleotide and detection and labeled oligonucleotide (Probing and Tagging Oligonucleotide；PTO) accordingly, capture
Oligonucleotides and capture and templating oligonucleotides (Capturing and Templating Oligonucleotide；CTO)
Accordingly, third oligonucleotides respectively with signal transduction oligonucleotides (Signaling Oligonucleotide；SO) or hybridization is few
Nucleotide (Hybridization Oligonucleotide；HO) corresponding.Signal transduction oligonucleotides, is caught at hybridization oligonucleotide
Catching can play the role of detecting oligonucleotides with templating oligonucleotides, extended chain or their combination.
The signal of the dimer formed in a manner of the cutting dependent on above-mentioned mediation oligonucleotides includes by rely on
In the letter that the dimer that the mode of the cutting of above-mentioned mediation oligonucleotides is formed inhibits the mode of the formation of other dimers to provide
Number (for example, detection and labeled oligonucleotide cutting and the non-hybridization of extension-dependence).
For example, generating when by detection and labeled oligonucleotide cutting and extension method based on to depend on above-mentioned mediation few
When the signal for the dimer that the mode of the cutting of nucleotide is formed, above-mentioned signal-generation mechanism includes: upstream oligonucleotide；It visits
It surveys and labeled oligonucleotide (Probing and Tagging Oligonucleotide；PTO), comprising mutual with target nucleic acid sequence
The hybridizing nucleotide sequence of benefit；It captures and templating oligonucleotides (Capturing and Templating
Oligonucleotide；CTO)；And template-dependence nucleic acid polymerase, there are 5 '-nucleases.Above-mentioned detection and mark
Note oligonucleotides includes: (i) 3 '-targeting moiety includes the hybridizing nucleotide sequence complementary with above-mentioned target nucleic acid sequence；And
(ii) 5 '-label position, comprising with the non-complementary nucleotide sequence of above-mentioned target nucleic acid sequence.Above-mentioned capture and templating widow's core
Thuja acid includes to capture position (i) and templating position (ii) along 3 ' → 5 ' directions, and above-mentioned capture position (i) includes and above-mentioned spy
Survey the nucleotide sequence with a part of complementation at 5 '-label positions of labeled oligonucleotide or 5 '-label positions, above-mentioned templating
Position (ii) includes 5 '-label positions and the non-complementary nucleotide of 3 '-targeting moieties with above-mentioned detection and labeled oligonucleotide
Sequence.
The specific embodiment of generation signal based on detection and labeled oligonucleotide cutting and extension method includes following step
Suddenly.
Include: step (a), hybridizes above-mentioned target nucleic acid sequence with upstream primer and detection and labeled oligonucleotide；Step
(b), under conditions of cutting above-mentioned detection and labeled oligonucleotide, making the result object of above-mentioned steps (a) and there are 5 ' cores
The template of phytase activity-dependence nucleic acid polymerase is in contact, and above-mentioned upstream oligonucleotide or the induction of its extended chain are based on having
The above-mentioned detection of the above-mentioned enzyme of 5 ' nucleases and the cutting of labeled oligonucleotide, above-mentioned cutting release comprising above-mentioned detection and
The 5 ' of labeled oligonucleotide-mark the segment of a part at position or 5 '-label positions；Step (c) makes from above-mentioned detection and mark
The above-mentioned segment and capture and templating oligonucleotide hybridization that oligonucleotides is released are remembered, from above-mentioned capture and templating oligonucleotides
The above-mentioned segment released hybridizes with the capture position of above-mentioned capture and templating oligonucleotides；Step (d) utilizes above-mentioned steps
(c) result object and template-dependence nucleic acid polymerase carries out extension, by making and above-mentioned capture and templating few nucleosides
The above-mentioned segment of the above-mentioned capture position hybridization of acid extends to form extension dimer, and above-mentioned extension dimer has and can pass through
(i) sequence and/or length or (III) of the sequence of above-mentioned segment and/or length, (ii) above-mentioned capture and templating oligonucleotides
The Tm value of the sequence and/or length adjustment of the sequence and/or length and above-mentioned capture and templating oligonucleotides of above-mentioned segment, on
It states and extends at least one mark that dimer is connected according to (i) with above-mentioned segment and/or above-mentioned capture and templating oligonucleotides
Note, (ii) are inserted in label in above-mentioned extension dimer, (III) during above-mentioned extension during above-mentioned extension
The label that is inserted in above-mentioned extension dimer and it is connected with above-mentioned segment and/or above-mentioned capture and templating oligonucleotides
At least one label or (IV) insertion label (intercalating label) provide target signal；Step (e), above-mentioned extension dimerization
Object maintain its two chain form it is specified at a temperature of measure above-mentioned target signal, thus detect above-mentioned extension dimer, it is above-mentioned to prolong
The presence for stretching dimer indicates the presence of above-mentioned target nucleic acid sequence.In this case, in above-mentioned detection and labeled oligonucleotide
It further include denaturation between iterative cycles, to implement above-mentioned steps (a)~step (e) repeatedly in cutting and the method extended
All or part of.
In sentence " including denaturation between iterative cycles ", term " denaturation (denaturation) " means double-strand
Nucleic acid molecules are dissociated into single stranded nucleic acid molecule.
In above-mentioned detection and labeled oligonucleotide cutting and the step of extension method in (a), above-mentioned upstream few nucleosides are substituted
The primer sets for amplifying target nucleic acid sequence can be used in acid.In this case, it is cut in above-mentioned detection and labeled oligonucleotide
Further include denaturation between iterative cycles and in extension method, come implement repeatedly above-mentioned steps (a)~step (e) whole or
A part.
Make with capture and templating oligonucleotide hybridization detection and labeled oligonucleotide segment extend and formed extension
After chain, above-mentioned detection and labeled oligonucleotide cutting and extension method can be classified as the technique for detecting above-mentioned extended chain.Above-mentioned spy
It surveys and labeled oligonucleotide is cut and extension method is characterized in that, utilize above-mentioned extended chain and above-mentioned capture and templating widow's core
The formation of the above-mentioned extended chain of dimerization analyte detection between thuja acid.
There is also detect that above-mentioned extended chain formed other close to method.For example, available miscellaneous with above-mentioned extended chain specificity
The oligonucleotides of friendship, to detect the formation of above-mentioned extended chain (for example, detection and labeled oligonucleotide cutting and extension-dependence
Signal transduction oligonucleotide hybridization method).In this approach, signal can be connected to miscellaneous with above-mentioned extended chain specificity from (i)
The label of the oligonucleotides of friendship, (ii) be connected to the label of the oligonucleotides of above-mentioned extended chain specific hybrid and with above-mentioned spy
Label, (iii) that survey and labeled oligonucleotide segment are connected are connected to the oligonucleotides with above-mentioned extended chain specific hybrid
Label and the label being inserted in above-mentioned extended chain during above-mentioned extension or (iv) are connected to and above-mentioned extended chain is special
The label and intercalative dye of the oligonucleotides of specific hybridization provide.With selecting a property, signal can be connected from (i) with above-mentioned extended chain
Label or (ii) intercalative dye provide.
It with selecting a property, can be with above-mentioned capture and templating oligonucleotides and above-mentioned capture and template for inhibition by detection
Change the other methods of the hybridization between the oligonucleotides of oligonucleotides specific hybrid (for example, detection and labeled oligonucleotide are cut
Cut and the non-hybridizing method of extension-dependence) the above-mentioned extended chain of detection formed.This inhibition can be considered as and be used to show target nucleic acid sequence
What is arranged is existing.Above-mentioned signal can be connected to from (i) can be with the oligonucleotides of above-mentioned capture and templating oligonucleotide hybridization
Label, (iii) that label, (ii) are connected with above-mentioned capture and templating oligonucleotides are connected to can be with above-mentioned capture and template
The label or (iv) changing the label of the oligonucleotides of oligonucleotide hybridization and being connected with above-mentioned capture and templating oligonucleotides
Intercalative dye provides.
According to an example, can have with the above-mentioned oligonucleotides of above-mentioned capture and templating oligonucleotides specific hybrid with
The sequence (overlapping sequence) of above-mentioned detection and the overlapping of labeled oligonucleotide segment.
According to an example, above-mentioned detection oligonucleotides includes: can be with the oligonucleotides (example of above-mentioned extended chain specific hybrid
Such as, detection and labeled oligonucleotide cutting and extension-dependent signals transduction oligonucleotide hybridization method) and can be with above-mentioned capture
Oligonucleotides with templating oligonucleotides specific hybrid is (for example, detection and labeled oligonucleotide cutting and extension-dependence
Non- hybridizing method).According to the present invention, above-mentioned detection oligonucleotides includes the extended chain generated during reaction or capture and template
Change oligonucleotides.
Generally, based on detection and labeled oligonucleotide cutting and the method extended with the presence for depending on target nucleic acid sequence
Extended chain formation.In the present invention in order to include for provide a variety of methods of signal using above-mentioned term " based on detection
The method cut and extended with labeled oligonucleotide ", above-mentioned a variety of methods include by detecting the cutting with labeled oligonucleotide
And the step of extending to form extended chain.
It is cut based on detection and labeled oligonucleotide and the example of the method for extension generation signal includes the following steps, namely,
Include: step (a), hybridizes above-mentioned target nucleic acid sequence with upstream primer and detection and labeled oligonucleotide；Step (b), with
Under conditions of above-mentioned detection and the cutting of labeled oligonucleotide, makes the result object of above-mentioned steps (a) and there are 5 ' enzymatically active nucleic acids
Property enzyme be in contact, above-mentioned upstream oligonucleotide or its extended chain induction based on 5 ' nucleases above-mentioned enzyme it is upper
Detection and the cutting of labeled oligonucleotide are stated, the 5 '-labeling sections comprising above-mentioned detection and labeled oligonucleotide are released in above-mentioned cutting
The segment of a part of position or 5 '-label positions；Step (c) makes the sheet above released from above-mentioned detection and labeled oligonucleotide
Section and capture and templating oligonucleotide hybridization, the above-mentioned segment released from above-mentioned capture and templating oligonucleotides are caught with above-mentioned
It catches and hybridizes with the capture position of templating oligonucleotides；Step (d) utilizes the result object and template-dependence of above-mentioned steps (c)
Nucleic acid polymerase carries out extension, the above-mentioned segment hybridized with the above-mentioned capture position of above-mentioned capture and templating oligonucleotides
It is extended, to form extension dimer；Step (e), by detecting the signal there are dependence generated with above-mentioned extended chain
To detect the formation of above-mentioned extended chain.In above-mentioned steps (a), substituting above-mentioned upstream oligonucleotide be can be used for expanding target nucleus
The primer sets of acid sequence, in this case, in above-mentioned detection and labeled oligonucleotide cutting and the method extended, repeatedly
Further include between circulation denaturation, come implement repeatedly above-mentioned steps (a)~step (e) all or part of.
According to an example, based on dimer formation generate above-mentioned signal include based on above-mentioned dimer hybridization (for example,
The hybridization of dimer itself or the hybridization of third oligonucleotides) signal that is induced or based on inhibiting to cause because of dimer formation
Third oligonucleotides hybridization and the signal that is induced.
According to an example, signal-generation mechanism for each above-mentioned target nucleic acid sequence be based on dependent on it is above-mentioned
The mode of the cutting of the mediation oligonucleotides of target nucleic acid sequence specific hybrid forms signal-generation mechanism of dimer.
According to an example, at least one is with the cutting dependent on detection oligonucleotides in 2 signal-generation mechanisms
Mode generates signal-generation mechanism of signal.
In particular, above-mentioned signal is based on above-mentioned detection widow core after hybridizing above-mentioned detection oligonucleotides with target nucleic acid sequence
The cutting of thuja acid generates.
After hybridizing above-mentioned detection oligonucleotides with target nucleic acid sequence, the cutting based on above-mentioned detection oligonucleotides it is upper
Stating signal can be by including the more of TaqMan probe method (U.S. Patent No. 5210015 and U.S. Patent No. 5538848)
Kind method generates.
When generating above-mentioned signal by TaqMan probe method, above-mentioned signal-generation mechanism includes for expanding target nucleus
The primer sets of acid sequence, the TaqMan probe with label (for example, interaction property double labeling) appropriate and there are 5 '-cores
The nucleic acid polymerase of phytase activity.During target amplification, the TaqMan probe hybridized with above-mentioned target nucleic acid sequence is cut, and is produced
It is raw to indicate signal existing for above-mentioned target nucleic acid sequence.
It is included the following steps, namely by the particular case that TaqMan probe method generates signal, comprising: step (a) makes
Target nucleic acid sequence is stated to hybridize with the TaqMan probe with primer sets and label appropriate (for example, interaction property double labeling)
The step of；Step (b), the result object using above-mentioned steps (a) and the nucleic acid polymerase with 5 ' nucleases, to expand target
Nucleic acid sequence, above-mentioned TaqMan probe are released above-mentioned label by cutting；And step (c), from the above-mentioned label inspection released
Signal is surveyed to generate.
In particular, above-mentioned signal is with the cutting dependent on the mediation oligonucleotides with above-mentioned target nucleic acid sequence specific hybrid
Mode is generated by the cutting of detection oligonucleotides.
An example according to the present invention releases segment by cutting in the mediation oligonucleotides hybridized with target nucleic acid sequence
In the case of, above-mentioned segment and detection oligonucleotides specific hybrid, above-mentioned segment induce the cutting of above-mentioned detection oligonucleotides.
An example according to the present invention releases segment by cutting in the mediation oligonucleotides hybridized with target nucleic acid sequence
In the case of, above-mentioned segment is extended and cuts the detection few nucleosides comprising the hybridizing nucleotide sequence complementary with oligonucleotides is captured
Acid.
The signal of cutting based on the detection oligonucleotides in a manner of the cutting dependent on above-mentioned mediation oligonucleotides can
By including Invader analysis (U.S. Patent No. 5691142), detection and labeled oligonucleotide cutting and extension-dependence
Cut (PTO Cleavage and Extension-Dependent Cleavage；PCEC) method (WO 2012/134195)
And a variety of methods such as method recorded in U.S. Patent No. 7309573 generate.In particular, at U.S. Patent No. 7309573
The method of middle record can be considered as using the generation signal based on cutting based on detection and labeled oligonucleotide cutting and extension
One of method.In above-mentioned detection and labeled oligonucleotide cutting and the method extended, detection is based on above-mentioned extended chain
Formation the cutting with above-mentioned capture and the oligonucleotides of templating oligonucleotides specific hybrid, to can detect above-mentioned prolong
Stretch the formation of chain.In Invader analysis, segment is formed by the cutting of mediation oligonucleotides, and extended with no segment
Mode induces continuous cleavage reaction.
An example according to the present invention, when generating above-mentioned signal in a manner of the cutting dependent on detection oligonucleotides,
The cutting inducement signal of above-mentioned detection oligonucleotides changes or induces the releasing of marked segment to be detected.
When the formation of cutting and dimer of the signal-generation mechanism based on detection oligonucleotides generates signal simultaneously,
As long as above-mentioned signal-generation mechanism is based on cutting and generates signal, so that it may which machine occurs for the signal for being considered as providing signal based on cutting
Structure.
According to an example, the signal in a manner of the cutting dependent on above-mentioned detection oligonucleotides generates opposite for having
Compared with the target nucleic acid sequence of the Supreme People's Procuratorate's testing temperature.When the cutting based on above-mentioned detection oligonucleotides generates above-mentioned signal, even if in office
What also can detect the label released based on cutting at a temperature of.Therefore, the signal that the cutting based on above-mentioned detection oligonucleotides generates
It can be without using in the target nucleic acid sequence of the above-mentioned relatively low detection temperature with the detection temperature for needing to limit to.
According to an example, the signal generation of the cutting dependent on above-mentioned detection oligonucleotides is only used for a target nucleic acid sequence
Column.It, can not be in the case where the signal generation dependent on the cutting of above-mentioned detection oligonucleotides is used in 2 target nucleic acid sequences
Above-mentioned 2 target nucleic acid sequences are detected according to the mode that detection temperature is distinguished.
An example according to the present invention, for signal-production of the target nucleic acid sequence with above-mentioned relatively high detection temperature
Life structure is signal-generation mechanism of the cutting based on detection oligonucleotides, for above-mentioned relatively low detection temperature
Signal-generation mechanism of target nucleic acid sequence is the signal-generation mechanism formed based on dimer.
An example according to the present invention, for signal-production of the target nucleic acid sequence with above-mentioned relatively high detection temperature
Life structure is signal-generation mechanism of the cutting based on detection oligonucleotides, for above-mentioned relatively low detection temperature
Signal-generation mechanism of target nucleic acid sequence is to utilize dependent on the mediation few nucleosides with above-mentioned target nucleic acid sequence specific hybrid
The mode of the cutting of acid forms signal-generation mechanism of dimer.
According to an example, above-mentioned detection oligonucleotides is marked comprising at least one.
An example according to the present invention, above-mentioned detection oligonucleotides can be formed by least one oligonucleotides.According to the present invention
An example, when above-mentioned detection oligonucleotides is formed by multiple oligonucleotides, above-mentioned detection oligonucleotides can be with a variety of sides
Formula has label.For example, one in multiple oligonucleotides can have at least one label, multiple oligonucleotides can have to
One position of few 1 label or oligonucleotides can have at least one label and other positions can not have label.
The signal generated by above-mentioned 2 signals-generation mechanism is not distinguished by the detector of single type.Above-mentioned term
" not by the other signal in the detector area of single type " means the identical or substantially the same characteristics of signals (example because of them
Such as, optical characteristics, emission wavelength and electric signal) it is not mutually distinguishable by the detector of single type.For example, for 2 target nucleic acids
Sequence using it is identical label (for example, FAM) and in order to detect from the emission wavelength of FAM use single type detector when,
Then signal can not be detected otherwise with area.
Term " signal (single type of signal) of single type " used in the present specification means
The signal of identical or substantially identical characteristics of signals (for example, optical characteristics, emission wavelength and electric signal) is provided.For example, FAM and
CAL Fluor 610 provides different types of signal.
Term " detector (single type of detector) of single type " meaning used in the present specification
Taste for single type signal testing agency.Include signal for multiple and different types multiple channels (for example,
Photodiode) detector in, each channel (for example, photodiode) is equivalent to " detector of single type ".
An example according to the present invention, above-mentioned 2 signal-generation mechanisms include identical label, from the letter of above-mentioned label
It is not distinguished by the detector of above-mentioned single type number not.
In the present invention, useful label includes in a variety of labels well known in the art.For example, in the present invention
In, useful label include it is single labelled, interaction property double labeling, intercalative dye and insertion label (incorporating
label)。
For example, above-mentioned single labelled including fluorescent marker, luminescent marking, chemiluminescent labeling, electrochemical label and metal
Label.It is above-mentioned single labelled according to whether being present in double-strand or whether being present in single-stranded different to provide according to an example
Signal (for example, different signal strengths).It is above-mentioned single labelled for fluorescent marker according to an example.It is used in the present invention
Single fluorescent marker preferred kind and binding site be disclosed in U.S. Patent No. 7537886 and U.S. Patent No.
In No. 7348141, and its relevant teachings item includes to be used as reference in the present specification.For example, above-mentioned single fluorescent marker packet
Containing JOE, FAM, TAMRA, ROX and based on the label of fluorescein.Above-mentioned single fluorescent marker can pass through a variety of methods and few nucleosides
Acid is connected.For example, above-mentioned label can be by the inclusion of the septal area of carbon atom (for example, 3- carbon spacer region, 6- carbon spacer region or 12-
Carbon spacer region) it is connected with probe.
As the representative example of interaction property tagging system, fluorescence resonance energy transfer (FRET, fluorescence
Resonance energy transfer) tagging system includes fluorescent reporter (donor molecule) and quenching molecules (receptor point
Son).In fluorescence resonance energy transfer, energy donor can be fluorescence, and energy acceptor can be fluorescence or non-fluorescence
Property.In another form of interaction property tagging system, energy donor is non-fluorescence, for example, chromophore
(chromophore), energy acceptor is fluorescence.In another form of interaction property tagging system, energy acceptor is hair
Photosensitiveness (luminescent), for example, bioluminescence, chemiluminescence or electrochemistry reflective, receptor are fluorescence.It is above-mentioned
Interaction property tagging system includes the dual mark based on " contact-mediation quenching (contact-mediated quenching) "
Note (, the Nucleic Acids such as Salvatore Research, 2002 (30) no.21e122 and Johansson etc.,
J.AM.CHEM.SOC 2002(124)pp 6950-6956).Interaction property tagging system also includes based at least two molecule
Any tagging system of interaction inducement signal variation between (for example, dyestuff).
In the present invention, useful reporter molecule and quenching molecules may include well known in the technical field of the invention
Any molecule.Concrete example is as follows: Cy2TM(506)、YO-PROTM-1(509)、YOYOTM-1(509)、Calcein(517)、FITC
(518)、FluorXTM(519)、AlexaTM(520)、Rhodamine 110(520)、Oregon GreenTM500(522)、
Oregon GreenTM488(524)、RiboGreenTM(525)、Rhodamine GreenTM(527)、Rhodamine 123
(529)、Magnesium GreenTM(531)、Calcium GreenTM(533)、TO-PROTM-1(533)、TOTO1(533)、
JOE(548)、BODIPY530/550(550)、Dil(565)、BODIPY TMR(568)、BODIPY558/568(568)、
BODIPY564/570(570)、Cy3TM(570)、AlexaTM546(570)、TRITC(572)、Magnesium OrangeTM
(575)、Phycoerythrin R&B(575)、Rhodamine Phalloidin(575)、Calcium OrangeTM(576)、
Pyronin Y(580)、Rhodamine B(580)、TAMRA(582)、Rhodamine RedTM(590)、Cy3.5TM(596)、
ROX(608)、Calcium CrimsonTM(615)、AlexaTM594(615)、Texas Red(615)、Nile Red(628)、
YO-PROTM-3(631)、YOYOTM-3(631)、R-phycocyanin(642)、C-Phycocyanin(648)、TO-PROTM-3
(660)、TOTO3(660)、DiD DilC(5)(665)、Cy5TM(670)、Thiadicarbocyanine(671)、Cy5.5
(694)、HEX(556)、TET(536)、Biosearch Blue(447)、CAL Fluor Gold 540(544)、CAL Fluor
Orange560(559)、CAL Fluor Red 590(591)、CAL Fluor Red 610(610)、CAL Fluor Red
635(637)、FAM(520)、Fluorescein(520)、Fluorescein-C3(520)、Pulsar 650(566)、Quasar
570 (667), Quasar 670 (705) and Quasar 705 (610).Number in bracket is to be indicated most as unit of nanometer
Big emission wavelength.Preferably, reporter molecule-quenching molecules include JOE, FAM, TAMRA, ROX and the label based on fluorescein.
Suitable fluorescent molecule and suitable report-quenching pair: Pesce etc. are disclosed in following various kinds of document,
editors,Fluorescence Spectroscopy(Marcel Dekker,New York,1971)；White etc.,
Fluorescence Analysis:A Practical Approach(Marcel Dekker,New York,1970)；
Berlman,Handbook of Fluorescence Spectra of Aromatic Molecules,2nd Edition
(Academic Press,New York,1971)；Griffiths,Color AND Constitution of Organic
Molecules(Academic Press,New York,1976)；Bishop,editor,Indicators(Pergamon
Press,Oxford,1972)；Haugland,Handbook of Fluorescent Probes and Research
Chemicals(Molecular Probes,Eugene,1992)；Pringsheim,Fluorescence and
Phosphorescence(Interscience Publishers,New York,1949)；Hauglandd,R.P.,
Handbook of Fluore 9scent Probes and Research Chemicals,6th EditionMolecular
Probes,Eugene,Oreg.,1996)；U.S. Patent No. No. 3996345 and No. 4351760.
In the present invention, it is worth noting that, using be capable of the fluorescence of wavelength or specific wavelength to a variety of ranges into
The non-fluorescent quenching molecule (for example, black quenching molecules or dark quencher) of row quenching.
In the signal transduction system comprising reporter molecule and quenching molecules, above-mentioned reporter molecule includes fluorescence resonance energy
The donor of transfer, quenching molecules include another partner (receptor) of fluorescence resonance energy transfer.For example, by fluorescein(e) dye
(fluorescein dye) is used as reporter molecule, and rhodamine (rhodamine dye) is made as quenching molecules
With.
Above-mentioned interaction double labeling can be connected with any chain in dimer.When including above-mentioned phase interaction
With the chain of property double labeling with single-chain state in the presence of, above-mentioned chain induces phase interaction by forming hair clip or random coil structure
With the quenching between property double labeling.When above-mentioned chain forms dimer, above-mentioned quenching is reduced.With selecting a property, when above-mentioned mutual
When functionality double labeling is connected with the nucleotide for the position for being located at neighbouring chain, generate between interaction property double labeling
Quenching.In the case where above-mentioned chain forms dimer and cut, above-mentioned quenching is reduced.
Each interaction property double labeling can be connected with two chains of dimer respectively.The formation of above-mentioned dimer lures
Quenching is led, the deformation inductdion of above-mentioned dimer is quenched.With selecting a property, a case where chain in two chains is cut
Under, it can induce and quenched.
It include in the present invention SYBRTM Green I, PO-PROTM-1, BO- as the example of usefully intercalative dye
PROTM-1、SYTOTM43、SYTOTM44、SYTOTM45、SYTOXTMBlue、POPOTM-1、POPOTM-3、BOBOTM-1、
BOBOTM-3、LO-PROTM-1、JO-PROTM-1、YO-PROTM1、YO-PROTM1、TO-PROTM1、SYTOTM11、SYTOTM13、
SYTOTM15、SYTOTM16、SYTOTM20、SYTOTM23、TOTOTM-3、YOYOTM3、GelStarTMAnd thiazole orange (thiazole
orange).Above-mentioned intercalative dye is specifically inserted in double-stranded nucleic acid molecule and generates signal.
Generate signal process in can in a manner of being inserted into label between extended peroid using insertion label (for example,
Plexor method, Sherrill C B, etc. Journal of the American Chemical Society, 126:4550-
45569(2004)).Also, based on the shape in a manner of the cutting dependent on the mediation oligonucleotides hybridized with target nucleic acid sequence
At the signal of dimer generate process above-mentioned insertion can also be used to mark.
In general, above-mentioned insertion label can be combined with nucleotide.Also, also using with non-natural nucleotide base.
Term " non-natural base (non-natural base) " used in the present specification means that hydrogen can be formed
The natural base of adenine (A), guanine (G), thymidine (T), cytimidine (C) and the uracil (U) of key base-pair etc.
Derivative.Term " non-natural base " as used in the present invention includes as parent compound (mother compound)
Base with the base-pair mode different from natural base, for example, being disclosed in U.S. Patent No. 5432272, United States Patent (USP)
In No. 5965364, U.S. Patent No. 6001983 and U.S. Patent No. 6037120.Base between non-natural base
To same with trona base phase, there are 2 or 3 hydrogen bonds.Also, the also shape in a specific way of the base-pair between non-natural base
At.It include following base in base-pairing combinations in the particular case of non-natural base.iso-C/iso-G,iso-dC/iso-
DG, K/X, H/J and M/N (referring to U.S. Patent No. 7422850).
In the case where generating signal by detection and labeled oligonucleotide cutting and extension method, above-mentioned extension is being carried out
The above-mentioned nucleotide being inserted into during reaction can have the first non-natural base, and above-mentioned hybridization-capture and templating oligonucleotides can
With nucleotide, above-mentioned nucleotide has the second non-day that specific binding compatibility is generated to the above-mentioned first non-natural base
Right base.
Term " target nucleic acid ", " target nucleic acid sequence " or " target sequence " used in the present specification means to be detected
Or quantitative nucleic acid sequence.Above-mentioned target nucleic acid sequence not only includes double-strand, also comprising single-stranded.Above-mentioned target nucleic acid sequence not only includes
Newly-generated sequence in the reaction, the also sequence comprising being initially present in nucleic acid sample.
Above-mentioned target nucleic acid sequence includes all DNA (genomic deoxyribonucleic acid (gDNA) and complementary deoxidation
Ribonucleic acid (cDNA)) and ribonucleic acid molecule and their hybrid (chimeric nucleic acid).Above-mentioned sequence can be double-strand or list
Chain form.As parent material above-mentioned nucleic acid be double-strand in the case where, it is preferable that by above-mentioned two chains be prepared into it is single-stranded or
The single-stranded form in part.It include heating, alkali, formamide, urea and glyoxal processing, enzyme as the well known method for chain separation
Method (enzyme effect of such as untwisting) and conjugated protein, however, it is not limited to this.For example, chain separation can be in 80 DEG C~105 DEG C of temperature
It is heated in degree range to realize.In document [Joseph Sambrook, etc. Molecular Cloning, A
Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.
(2001)] conventional method for realizing this processing is disclosed in.
When messenger RNA (mRNA) is used as parent material, reverse transcription must be carried out before carrying out annealing steps
Step, related detailed content are disclosed in document [Joseph Sambrook, etc. Molecular Cloning, A
Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.
(2001)；And Noonan, K.F. etc., in Nucleic Acids Res.16:10366 (1988).In order to realize reverse transcription reaction,
Draw using oligonucleotides dT primer, random primer or the target-specific that can hybridize with the poly A tail of messenger RNA
Object.
Target nucleic acid sequence includes any natural (naturally occurring) prokaryotic cell nucleic acid, eukaryocyte (example
Such as, protozoan and parasitic animal, mushroom, yeast, higher plant, lower animal and high dynamic comprising mammal and the mankind
Object) nucleic acid, virus (for example, herpesviral, human immunodeficiency virus (HIV), influenza virus, Epstein-Barr virus, hepatitis virus and
Poliovirus etc.) nucleic acid or viroid nucleic acid.Also, above-mentioned nucleic acid molecules can be by recombinant production or can give birth to
Any nucleic acid molecules of production or chemical synthesis can chemically synthesized any nucleic acid molecules.Therefore, it can find or can not send out naturally
Existing above-mentioned nucleic acid sequence.Above-mentioned target nucleic acid sequence may include known or unknown sequence.
In the present specification term " sample " mean the cell from biology supply source, tissue, fluid or can according to this
Other any media (medium) that invention is valuably evaluated, and include virus, bacterium, tissue, cell, blood, serum,
Blood plasma, lymph, milk, urine, excrement, intraocular liquid, saliva, sperm, brain extract, spinal fluid, appendix, spleen and almond tissue
Extract, amniotic fluid, ascites and non-biological sample sample (for example, food and water).Also, said sample includes from biology
The nucleic acid molecules of supply source insulation and the nucleic acid molecules of synthesis.
An example according to the present invention implements above-mentioned steps (a) during with the amplification of signal of nucleic acid amplification.
In the present invention, the above-mentioned signal generated by signal-generation mechanism can be expanded with target and be expanded together.Select a property
Ground, above-mentioned signal can be expanded with expanding without target.
An example according to the present invention, above-mentioned signal generates and target amplification is real during comprising amplification of signal together
It applies.
An example according to the present invention, according to polymerase chain reaction (Polymerase chain reaction, PCR)
Implement above-mentioned target amplification.For target amplification, polymerase chain reaction is being utilized extensively in the technical field of the invention, it is above-mentioned poly-
Polymerase chain reaction includes the circulation of the annealing (hybridization) and primer extend between denaturation, target sequence and the primer of target sequence
(Mullis etc., U.S. Patent No. 468319, No. 4683202 and No. 4800159；Saiki etc., (1985) Science
230,1350-1354).Can by be applicable in during polymerase chain reaction above-mentioned signal generating method (for example,
TaqMan method and the method cut and extended based on detection and labeled oligonucleotide) come amplified signal.According to an example, this hair
It is bright to provide signal by real-time polymerase chain reaction method.According to an example, the amplification of above-mentioned target nucleic acid sequence passes through polymerization
Enzyme chain reaction (polymerase chain reaction；PCR), ligase chain reaction (ligase chain
reaction；LCR, referring to Wiedmann M, etc. " Ligase chain reaction (LCR)-overview and
applications."PCR Methods and Applications 1994Feb；3 (4): S51-64), notch fill up connection
Enzyme chain reaction (gap filling LCR；GLCR, referring to WO 90/01069, European Patent No. 439182 and WO 93/
00447), Q- β replicates enzymatic amplification (Q-beta replicase amplification；Q-beta, reference Cahill P, etc.,
Clin Chem., 37 (9): 1482-5 (1991), U.S. Patent No. 5556751), strand displacement amplification (strand
displacement amplification；SDA, referring to G T Walker etc., Nucleic Acids Res.20 (7):
16911696 (1992), European Patent No. 497272), amplification (the nucleic acid sequence- based on nucleic acid sequence
based amplification；NASBA, referring to Compton, J.Nature 350 (6313): 912 (1991)), transcriptive intermediate
Amplification (Transcription-Mediated Amplification；TMA, referring to Hofmann WP etc., J Clin
Virol.32(4):289-93(2005)；U.S. Patent No. 5888779) or rolling circle amplification (Rolling Circle
Amplification；RCA, referring to Hutchison C.A. etc., Proc.Natl Acad.Sci.USA.102:1733217336
(2005)) implement.
Above-mentioned amplification method can have not been changed a serial response of temperature by change temperature or repetition to expand target sequence
Column.Indicate to include the unit for repeating the amplification of an above-mentioned serial response with " circulation (cycle) ".According to the above-mentioned circulation of amplification method
Unit can be indicated with number of repetition or time.
For example, can be in endpoint (the end-point of of each a part circulation or reaction for recycling, selecting of amplification
Reaction signal detection is carried out in).It, can be all in the case where detecting signal at least two circulation according to an example
Implement the signal detection in each circulation at a temperature of detection or at a temperature of the detection of selection a part.According to the present invention one
Example carries out above-mentioned detection in the circulation of odd number at a temperature of relatively high detection, in the circulation of even number, relatively
Above-mentioned detection is carried out at a temperature of high detection.
Target amplification is can be achieved together with generating based on signal-generation mechanism signal in an example according to the present invention
Under the conditions of cultivated.
An example according to the present invention carries out above-mentioned steps (a) during the amplification of signal of free nucleic acid amplification.
By including the method generation signal of cutting of oligonucleotides, above-mentioned signal can be expanded with no target
Mode be amplified.Although for example, can by CPT method (Duck P, etc. Biotechniques, 9:142-148 (1990)),
Invader analyzes (U.S. Patent No. No. 6358691 and No. 6194149), based on detection and labeled oligonucleotide cutting and prolongs
The method stretched is (for example, detection and labeled oligonucleotide cutting and extension-dependent signals transduction oligonucleotide hybridization method, spy
Survey and labeled oligonucleotide cutting and the non-hybridizing method of extension-dependence and PCEC method) or according to CER method (WO 2011/
037306) make amplification of signal, but carry out above-mentioned steps (a) in a manner of not amplifying target nucleic acid sequence.
Above-mentioned amplification method can have not been changed a serial response of temperature by change temperature or repetition to expand target sequence
Column.Indicate to include the unit for repeating the amplification of an above-mentioned serial response with " circulation (cycle) ".According to the above-mentioned circulation of amplification method
Unit can be indicated with number of repetition or time.
For example, signal detection can be carried out in the endpoint of each a part circulation or reaction for recycling, selecting of amplification.
Above-mentioned target nucleic acid sequence is realized by the target amplification mechanism comprising primer sets and nucleic acid polymerase for amplification
Amplification.
An example according to the present invention, can be used has nuclease (for example, 5 ' nucleases or 3 ' enzymatically active nucleic acids
Property) nucleic acid polymerase.The nucleic acid polymerase without nuclease can be used in an example according to the present invention.
In the present invention, having useful nucleic acid polymerase is the thermal stability DNA obtained from various bacterium kinds
Polymerase, above-mentioned deoxyribonucleic acid polymerase include thermus aquaticus (Taq, Thermus aquaticus), Thermophilic Bacterium
(Tth, Thermus thermophilus), Filamentous Thermus (Thermus filiformis), yellow Thermus (Thermis
Flavus), super good hot archeobacteria (Thermococcus literalis), typical Thermophilic Bacteria (Thermus
Antranikianii), card moral Phallus Thermophilic Bacteria (Thermus caldophilus), Qie Laluo Phallus Thermophilic Bacteria
(Thermus chliarophilus), yellow Thermus (Thermus flavus), fire ground Thermus (Thermus
Igniterrae), thermophilic hedgehog fungus (Thermus lacteus), dead Thermophilic Bacteria (hermus oshimai), red Thermus
(Thermus ruber), Lu Bensi Thermus (Thermus rubens), thermus aquaticus (Thermus scotoductus),
Xi Fannasire robe (Thermus silvanus), Thermus kind Z05 (Thermus species Z05), Thermus kind sps
17 (Thermus species sps 17), Thermophilic Bacteria (Thermus thermophilus), Thermotoga maritima (hermotoga
Maritima), dwell thermobacillus (Thermotoga neapolitana), Africa of Naples is dwelt hot chamber bacterium (Thermosipho
Africanus), thermophilic coccus (Thermococcus litoralis), the western thermobacillus (Thermococcus of Barrow
Barossi), Pyrococcus furiosus (Thermococcus gorgonarius), Thermotoga maritima (Thermotoga
Maritima), dwell thermobacillus (Thermotoga neapolitana), Africa of Naples is dwelt hot chamber bacterium
(Thermosiphoafricanus), walsh hot-bulb bacterium (Pyrococcus woesei), Pyrococcus horikoshii (Pyrococcus
Horikoshii), P.abyssi (Pyrococcus abyssi), hidden heat supply network bacterium (Pyrodictium occultum), thermophilic
Fire produces liquid bacterium (Aquifex pyrophilus) and Pseudomonas hyperthermophile (Aquifex aeolieus).In particular instances,
Thermal stability deoxyribonucleic acid polymerase is Taq polymerase.
An example according to the present invention is realized above-mentioned by asymmetry polymerase chain reaction (asymmetric PCR)
Target nucleic acid sequence amplification.The ratio of above-mentioned primer can be selected by considering cutting or the hybridization of downstream oligonucleotide.
Together with 2 signal-generation mechanisms for generating signal, during the culture (reaction) of sample or after culture,
Generated signal is detected using the detector of single type.
One in above-mentioned 2 target nucleic acid sequences has by corresponding above-mentioned signal-generation mechanism determination relatively
The Supreme People's Procuratorate's testing temperature, another has the relatively low detection temperature determined by corresponding above-mentioned signal-generation mechanism.
" target nucleic acid sequence has true by corresponding above-mentioned signal-generation mechanism for expression used in the present specification
Fixed detection temperature " means to detect target nucleic acid sequence at a temperature of having the detection for being pre-allocated in above-mentioned target nucleic acid sequence
Column, above-mentioned detection temperature can detect that from the signal-generation mechanism designed in a manner of generating signal at a temperature of above-mentioned detection
The signal detection of generation.
An example according to the present invention is divided by the detection temperature that corresponding above-mentioned signal-generation mechanism determines
Fit over a target nucleic acid sequence.
Above-mentioned relatively high detection temperature is that can produce for the target nucleic acid sequence with above-mentioned relatively high detection temperature
Signal temperature, it is above-mentioned it is relatively low detection temperature be can produce for it is above-mentioned it is relatively low detection temperature target nucleic acid
The signal of sequence and for it is above-mentioned it is relatively high detection temperature target nucleic acid sequence signal.
One of the features of the present invention is that detection indicates signal existing for 2 target nucleic acid sequences at a temperature of different detections, from
And it is determined by the presence of 2 target nucleic acid sequences of difference.
According to an example, consider that the temperature range of signal can be generated by above-mentioned signal-generation mechanism to predefine needle
To the detection temperature of above-mentioned target nucleic acid sequence.
In the present invention, by the presence of the specific temperature that can generate signal in the way of dependent on signal-generation mechanism
The technical characteristic of range.
For example, signal-generation mechanism generates signal as 2 making nucleic acid molecular hybridizations (or combine), when non-hybridization (or solution
From) when, above-mentioned signal-generation mechanism does not generate signal, in the case, although the hybridization of 2 nucleic acid molecules can be achieved
At a temperature of generate signal, but do not generate signal at a temperature of 2 nucleic acid molecules are not hybridized.As described above, existing can
It generates the specific range of temperatures of signal (that is, signal detection) and does not generate other temperature ranges of signal.Above-mentioned temperature range by
The influence of the Tm value of the hybrid of 2 nucleic acid molecules used in above-mentioned signal-generation mechanism.
When using signal generating method, above-mentioned signal generating method is using having markd released piece after cutting
Section, theoretically, can detect signal under any temperature (for example, 30~99 DEG C).
It detects temperature and is selected from the temperature range that can generate signal by above-mentioned signal generating mechanism.
In the present invention, term " detection temperature range " is to especially describe and can produce signal generation (that is, signal is examined
Survey) temperature range and use.
It, there are when different detection temperature ranges, may be selected when according to signal-generation mechanism for 2 target nucleic acid sequences
Nonoverlapping detection temperature range (non-overlapped detection temperature range) is used as relatively high
Detect temperature.It is confirmed as having by the signal-generation mechanism detection target nucleic acid sequence for providing relatively high detection temperature
Above-mentioned relatively high detection temperature.Detection temperature range (the overlapped detection of overlapping may be selected
Temperature range) it is used for relatively low detection temperature.By the relatively low detection temperature of offer and do not provide relatively
The target nucleic acid sequence that signal-generation mechanism of the Supreme People's Procuratorate's testing temperature is detected is confirmed as having above-mentioned relatively low detection temperature
Target nucleic acid sequence.
According to an example, the position of above-mentioned nonoverlapping position and overlapping can be distinguished in a manner of not distinguishing mutually.Example
Such as, it at a temperature of the relatively high detection selected for the target nucleic acid sequence with above-mentioned relatively high detection temperature, is based on
The signal that target nucleic acid sequence with above-mentioned relatively low detection temperature provides can be generated with lower intensity.In such case
Under, it can be by properly selecting the conspicuousness for determining the signal detected at a temperature of above-mentioned relatively high detection
(significance) a reference value (reference value) come overcome because at a temperature of above-mentioned relatively high detection based on tool
Error signal (false signal) caused by the signal for having the target nucleic acid sequence of above-mentioned relatively high detection temperature to provide is asked
Topic.
It, can be by considering nonoverlapping detection temperature range and the detection temperature of overlapping in detection temperature according to an example
Range predefines above-mentioned detection temperature.
According to an example, for the above-mentioned detection temperature that target nucleic acid sequence is assigned mutually have at least 2 DEG C, 3 DEG C, 4 DEG C,
5 DEG C, 7 DEG C, 8 DEG C, 9 DEG C, 10 DEG C, 11 DEG C, 12 DEG C, 15 DEG C or 20 DEG C of difference.
According to the present invention, the presence for detecting each target nucleic acid sequence can be distributed by considering signal-generation mechanism
Temperature.
An example according to the present invention, one in above-mentioned 2 target nucleic acid sequences is allocated to relatively high detection temperature,
And another is allocated to relatively low detection temperature, after this, constructs the signal-generation machine for being suitable for above-mentioned detection temperature
Then structure carries out above-mentioned steps (a).
According to an example, above-mentioned relatively high detection temperature for being detected and above-mentioned relatively low can be predefined
Detect temperature.For example, above-mentioned relatively high detection temperature and above-mentioned relatively low detection temperature are redefined for 72 DEG C and 60
After DEG C, the signal-generation mechanism for being suitable for above-mentioned detection temperature is constructed, then carries out above-mentioned steps (a).
According to an example, firstly, distribution is for above-mentioned after constructing signal-generation mechanism for 2 target nucleic acid sequences
Then the detection temperature of 2 target nucleic acid sequences carries out above-mentioned steps (a).
An example according to the present invention, when above-mentioned signal-generation mechanism generates letter in a manner of being formed dependent on dimer
Number when, Tm based on above-mentioned dimer selects above-mentioned detection temperature.
An example according to the present invention, when above-mentioned signal-generation mechanism generates letter in a manner of being formed dependent on dimer
Number when, above-mentioned detection temperature can be adjusted by adjusting the Tm value of above-mentioned dimer.
For example, based on the detection oligonucleotides with above-mentioned target nucleic acid sequence specific hybrid (for example, LUX probe, molecule
Beacon probe, HyBecon probe and adjacent hybridization probe) generate above-mentioned signal in the case where, pass through and adjust above-mentioned dimer
Tm value, successfully to realize the detection of above-mentioned signal at predetermined temperature.When using scorpion primer, pass through adjusting
The Tm value at the position hybridized with extended chain, successfully to realize the detection of above-mentioned signal at predetermined temperature.
Based on the dimer formed according to the presence of above-mentioned target nucleic acid sequence to generate above-mentioned signal, pass through
The Tm value of above-mentioned dimer is adjusted, successfully to realize the detection of above-mentioned signal at predetermined temperature.For example, passing through
In the case that detection and labeled oligonucleotide cutting and extension method generate above-mentioned signal, in capture and templating oligonucleotides
By adjusting the Tm value of the extension dimer extended to form by detection and labeled oligonucleotide segment, predetermined
At a temperature of successfully realize the detection of above-mentioned signal.
It is above-mentioned that easily adjustable above-mentioned dimer is had based on detection and labeled oligonucleotide cutting and the method extended
Tm value or based on above-mentioned dimer on hybridization play influence third hybrid Tm value the advantages of.
An example according to the present invention, when above-mentioned signal-generation mechanism is with the side of the cutting dependent on detection oligonucleotides
When formula generates signal, above-mentioned detection temperature is arbitrarily selected.As long as that is, the letter that the detectable cutting based on detection oligonucleotides generates
Number, so that it may select any temperature.As described above, above-mentioned when being generated in a manner of the cutting dependent on above-mentioned detection oligonucleotides
When signal, the label released by above-mentioned cutting can be detected at various temperatures.
According to an example, when generating above-mentioned signal in a manner of the cutting dependent on above-mentioned detection oligonucleotides, can incite somebody to action
Above-mentioned detection temperature is used as opposite highest detection temperature.
As described above, by considering to depend on signal-generation mechanism detection temperature range, to determine above-mentioned detection temperature
Degree.Therefore, the above-mentioned signal detection of explanation that can be following at a temperature of particular detection, that is, at a temperature of relatively high detection
Above-mentioned detection is used to detect the target nucleic acid sequence with above-mentioned relatively high detection temperature, upper at a temperature of relatively low detection
Detection is stated for detecting the target nucleic acid sequence with above-mentioned relatively low detection temperature and there is above-mentioned relatively high detection temperature
The target nucleic acid sequence of degree.
For example, when the signal for the target nucleic acid sequence with above-mentioned relatively low detection temperature and for above-mentioned phase
When passing through detection and labeled oligonucleotide cutting and extension method generation to the signal of the target nucleic acid sequence compared with the Supreme People's Procuratorate's testing temperature,
It is generated based on the extension dimer with the Tm value for being suitable for above-mentioned relatively high detection temperature above-mentioned relatively high for having
The signal for detecting the target nucleic acid sequence of temperature, based on the extension dimerization with the Tm value for being suitable for above-mentioned relatively low detection temperature
Object generates the signal for the target nucleic acid sequence with above-mentioned relatively high detection temperature.At a temperature of above-mentioned relatively high detection
In the case where detecting signal, there is the extension dimer for the Tm value for being suitable for above-mentioned relatively low detection temperature to be dissociated into list
Chain, therefore signal is not generated, it thus only detects for the target nucleic acid sequence with above-mentioned relatively high detection temperature.Above-mentioned
In the case where detecting signal at a temperature of relatively low detection, there is prolonging for the Tm value for being suitable for above-mentioned relatively high detection temperature
It stretches dimer and the extension dimer with the Tm value for being suitable for above-mentioned relatively low detection temperature all has dimer form, by
This detect for it is above-mentioned it is relatively low detection temperature target nucleic acid sequence signal and for it is above-mentioned relatively
The signal of the target nucleic acid sequence of the Supreme People's Procuratorate's testing temperature.
In one example, it generates when by TaqMan method for the target nucleic acid sequence with above-mentioned relatively high detection temperature
Signal and by detection and labeled oligonucleotide cutting and extension method generate for have above-mentioned relatively low detection temperature
Target nucleic acid sequence signal when, provide by the fluorescent marker released for the target with above-mentioned relatively high detection temperature
The signal of nucleic acid sequence, and by the extension dimer offer needle with the Tm value for being suitable for above-mentioned relatively low detection temperature
To the signal of the target nucleic acid sequence with above-mentioned relatively low detection temperature.Letter is detected at a temperature of above-mentioned relatively high detection
In the case where number, there is the extension dimer for the Tm value for being suitable for above-mentioned relatively low detection temperature to be dissociated into single-stranded, thus
Signal is not generated, is thus only detected from glimmering for having released for the target nucleic acid sequence with above-mentioned relatively high detection temperature
The signal of signal.In the case where detecting signal at a temperature of above-mentioned relatively low detection, not only detect suitable from having
In the signal that the extension dimer of the Tm value of above-mentioned relatively low detection temperature provides, also detect from the fluorescent marker of releasing
Signal is detected as a result, for the signal of the target nucleic acid sequence with above-mentioned relatively low detection temperature and above-mentioned for having
The signal of the target nucleic acid sequence of relatively high detection temperature..
Detector as used in the present invention includes any mechanism of detectable signal.For example, when using fluorescence signal
When, the photodiode for being suitable for detecting fluorescence signal can be used as detector.Utilize the detector of single type
Detection means the detector of the signal of detectable single type or utilizes the inspection comprising including a variety of channels (that is, photodiode)
Each channel (that is, photodiode) of device is surveyed to be detected.
According to an example, the generation of above-mentioned signal includes from label " generating or eliminate signal " and " increasing or decreasing letter
Number ".
Step (b): the presence of target nucleic acid sequence is determined
After detecting signal, the presence of 2 target nucleic acid sequences is determined by the signal detected in above-mentioned steps (a).
Determine that there is above-mentioned relatively high detection temperature by the signal detected at a temperature of above-mentioned relatively high detection
Target nucleic acid sequence presence.According to the signal detected at a temperature of above-mentioned relatively high detection and above-mentioned relatively low
Difference between the signal detected at a temperature of detection determines depositing for the target nucleic acid sequence with above-mentioned relatively low detection temperature
In.
The signal used to determine the presence of target include by the multi-signal characteristic obtained from signal detection into
Row Mathematical treatment come the value that obtains, for example, comprising to signal strength (for example, RFU (Relative fluorescence units) value, expand when
Particular cycle, the RFU value in the circulation of selection or endpoint), signal intensity shape (or pattern), CtValue or above-mentioned characteristic carry out
Mathematical treatment is come the value that obtains.
An example according to the present invention, in the case where obtaining amplification curve by real-time polymerase chain reaction, in order to
Determine that the multi-signal value (or characteristic) from above-mentioned amplification curve may be selected in the presence of target.
In order to determine have it is above-mentioned it is relatively high detection temperature target nucleic acid sequence presence may be used at it is above-mentioned relatively
Characteristic of signal obtained at a temperature of high detection itself.
With selecting a property, in order to determine the presence with above-mentioned relatively high detection temperature target nucleic acid sequence, it can be used and pass through
Mathematical treatment is carried out come the signal of the deformation provided to the characteristic of above-mentioned signal.
In order to which the difference obtained between the signal at a temperature of relatively high detection temperature and relatively low detection can be used
State the above-mentioned characteristics of signals itself at a temperature of characteristics of signals at a temperature of relatively high detection itself and relatively low detection.
With selecting a property, above-mentioned relatively high detection temperature and above-mentioned phase can be made by carrying out Mathematical treatment to characteristics of signals
To one or all deformation in the signal at a temperature of lower detection, and for obtaining relatively high detection temperature and relatively
The difference between signal at a temperature of low detection.
According to an example, with sentence " signal detected at a temperature of relatively high detection temperature and relatively low detection "
The term " signal " relatively used is embodied not only in the signal itself obtained at a temperature of detection, also comprising by above-mentioned signal
Mathematical treatment is carried out come the signal of the deformation provided.
According to an example, in the case where carrying out above-mentioned Mathematical treatment, the characteristic of above-mentioned signal should be that mathematics can be achieved
The characteristic of processing.In particular instances, above-mentioned Mathematical treatment including the use of signal calculating (for example, addition, multiplication, subtraction and
Division) or obtain from above-mentioned signal other values processing.In the present invention, in order to determine the presence of target nucleic acid sequence
And the signal used is usually significant signal (significant signal).That is, above-mentioned signal is to rely on target nucleic acid sequence
Presence and the signal that generates.On the other hand, when calculating the signal that detects at a temperature of above-mentioned relatively high detection and upper
When stating the difference between the signal detected at a temperature of relatively low detection, in order to calculate above-mentioned difference, background signal can be used
Deng the signal of no conspicuousness.As long as in connection with this, in order to which the signal for determining the presence of target nucleic acid sequence and using can be used in
It calculates difference or participates in determination process, so that it may which being understood as above-mentioned signal not only includes the signal with conspicuousness, also includes nothing
The signal of conspicuousness.
According to an example, the conspicuousness of the signal detected can be determined using threshold value.For example, to consider detector
After the mode of background signal, susceptibility or used label predefines threshold value from negative control group, it may be determined that from sample
The conspicuousness of signal.
In the case where detecting signal (that is, significant signal) at a temperature of relatively high detection, it is determined as existing with upper
State the target nucleic acid sequence of relatively high detection temperature.
Herein, the signal of no conspicuousness can be indicated with the sentence of " signal is not present " or " not detecting signal ".
In the present specification, " signal (by a is based on the term that the presence of target nucleic acid sequence relatively uses is determined
Signal) " mean by with utilize signal digital value or their deformation, using signal in the presence/absence of and ratio
The signal generated by signal-generation mechanism is directly or indirectly utilized compared with threshold value and the mode of signal or is made by signal-generation machine
The signal skew that structure generates determines the presence of target nucleic acid sequence.
In the present specification, it is relatively used with the presence of the determining target nucleic acid sequence with relatively high detection temperature
Term " determination (determination by a signal) signal-based " may include by considering in relatively high detection temperature
The conspicuousness of the signal detected under degree come determine with it is relatively high detection temperature target nucleic acid sequence existing mode.
In the present invention, according to the signal detected at a temperature of above-mentioned relatively high detection and in relatively low detection temperature
The signal detected under degree determines the presence with the target nucleic acid sequence of relatively low detection temperature.
In the case where detecting signal at a temperature of relatively low detection, can not be determined using above-mentioned signal itself with phase
Presence to the target nucleic acid sequence of lower detection temperature.It is directed to the reason for this is that can detect that at a temperature of above-mentioned relatively low detection
The signal of target nucleic acid sequence with relatively high detection temperature.
It is a feature of the present invention that in order to analyze the signal detected at a temperature of above-mentioned relatively low detection,
And utilize the signal detected at a temperature of above-mentioned relatively high detection.
Interestingly present inventors understand that arriving, in a reaction vessel, detected at a temperature of predetermined 2 detections
When out for indicating the existing signal of single target nucleic acid sequence, with specific pattern (rule), there are signal intensities.
For example, being examined at a temperature of relatively high detection for the target nucleic acid sequence with above-mentioned relatively high detection temperature
Signal intensity between the signal measured and the signal detected at a temperature of relatively low detection is in specific pattern (rule).Example
Such as, at a temperature of 2 detections, the intensity of above-mentioned signal can be identical or substantially the same, even if the intensity of above-mentioned signal is mutually not
Just as, but may be present in particular range.
It is a feature of the present invention that the above results to be suitable for the detection of target nucleic acid sequence.
In a reaction vessel, for the signal of target nucleic acid sequence only to detect the different mode of temperature (for example, without target
The variation of content or variation without buffer conditions) exist in the signal intensity that detects, thus detect between temperature at 2
Specific pattern (rule).Based on the specific pattern (rule) in signal intensity, in order to analyze in above-mentioned relatively low detection temperature
Under the signal that detects may be used at the signal detected at a temperature of above-mentioned relatively high detection.
According to an example, there are specific patterns in the signal intensity between 2 detection temperature for target nucleic acid sequence
Implement method of the invention under conditions of (rule).
According to an example, in order to confirm whether the signal detected at a temperature of above-mentioned relatively low detection includes by having
The signal that the target nucleic acid sequence of above-mentioned relatively low detection temperature provides, and utilize and detected at a temperature of above-mentioned relatively high detection
Signal out, and determined in a manner of analyzing the signal that detects at a temperature of above-mentioned relatively low detection have it is above-mentioned relatively
The presence of the target nucleic acid sequence of low detection temperature.
Can at a temperature of obtaining in above-mentioned relatively high detection the signal that detect and in above-mentioned relatively low detection temperature
Under after difference between the signal that detects, above-mentioned difference is analyzed, is thus implemented using in above-mentioned relatively the Supreme People's Procuratorate
The analysis for the signal that the mode of the signal detected under testing temperature detects at a temperature of above-mentioned relatively low detection.
An example according to the present invention can be obtained according to using the principle of the signal at a temperature of above-mentioned relatively high detection
The target nucleic acid sequence with above-mentioned relatively low detection temperature in the signal detected at a temperature of above-mentioned relatively low detection
The size (or part) of signal.
According to an example, by the signal detected at a temperature of above-mentioned relatively high detection and in above-mentioned relatively low inspection
Difference between the signal detected under testing temperature determines the presence with the target nucleic acid sequence of above-mentioned relatively low detection temperature.
For example, in the case where (i) in sample there is only with the relatively high target nucleic acid sequence for detecting temperature, opposite
Compared with detecting signal at a temperature of the Supreme People's Procuratorate's testing temperature and relatively low detection.It is detected at a temperature of above-mentioned relatively high detection
Signal, which exists, is possible to different from the signal detected at a temperature of above-mentioned relatively low detection.Due in addition to detection temperature it
Outside, a possibility that all conditions of this species diversity are identical, are consequently belonging in particular range is very high.What is calculated sample
In the case that above-mentioned difference belongs in particular range, the signal detected at a temperature of above-mentioned relatively low detection is only in tool
There is the target nucleic acid sequence of above-mentioned relatively high detection temperature.That is, can be identified as the target nucleus with above-mentioned relatively low detection temperature
Acid sequence is not present in sample.
(ii) there is the target nucleic acid sequence with relatively high detection temperature in sample and there is relatively low detection
In the case where the target nucleic acid sequence of temperature, signal is detected at a temperature of relatively high detection temperature and relatively low detection.
It, can due to there is the target nucleic acid sequence with above-mentioned relatively low detection temperature, thus compared with the difference in above-mentioned example (i)
Preferably distinguish the difference between above-mentioned signal.The target with above-mentioned relatively low detection temperature can be determined using above-mentioned difference
The presence of nucleic acid sequence.
(iii) in sample there is only in the case where the target nucleic acid sequence with relatively low detection temperature, although can be
Signal is detected at a temperature of relatively low detection, but does not detect signal at a temperature of relatively high detection.In relatively the Supreme People's Procuratorate
Do not detect that signal indicates that there is no the target nucleic acid sequences with above-mentioned relatively high detection temperature under testing temperature, thus above-mentioned
The signal detected at a temperature of relatively low detection can recognize as based on the target nucleic acid sequence with above-mentioned relatively low detection temperature
Column.Thus, it may be determined that the presence of the target nucleic acid sequence with above-mentioned relatively low detection temperature.
With selecting a property, aobvious using the nothing detected at a temperature of above-mentioned relatively high detection in above-mentioned example (iii)
The signal of work property (for example, background signal) obtains above-mentioned difference.In this alternative, above-mentioned difference and above-mentioned example
(i) a possibility that difference in is really different are very high, thus can determine the target nucleic acid with above-mentioned relatively low detection temperature
The presence of sequence.
It can be according to the difference between a variety of signals detected at a temperature of above-mentioned detection close to method acquisition.
In the present specification, it is relatively used with " based on the difference (or utilizing the difference between signal) between signal "
Term " difference (difference) " not only include that Mathematical treatment is carried out the difference that obtains to the signal of signal itself or deformation
It is different, but also including present or absent difference signal-based.For example, can be by calculating at a temperature of relatively high detection
Ratio (ratio) or subtraction between the signal detected and the signal detected at a temperature of relatively low detection
(subtraction) above-mentioned difference is obtained.With selecting a property, can make one detection at a temperature of signal skew, and by its with
It detects the signal under temperature at other to be compared, to obtain above-mentioned difference.It can be indicated at a variety of aspects (aspect)
Difference between the signal detected at a temperature of relatively high detection and the signal detected at a temperature of relatively low detection.
For example, can by with numerical value, signal in the presence/absence of or the drafting of characteristics of signals indicate above-mentioned difference.
An example according to the present invention, the signal detected at a temperature of above-mentioned relatively high detection and it is above-mentioned relatively
The difference between signal detected at a temperature of low detection includes to the signal detected at a temperature of above-mentioned relatively high detection
And the difference that the signal detected at a temperature of above-mentioned relatively low detection carries out Mathematical treatment to obtain.
An example according to the present invention considers in the case where not detecting signal at a temperature of above-mentioned relatively high detection
Signal is not detected at a temperature of above-mentioned relatively high detection, it can be based on the letter detected under above-mentioned relatively low testing temperature
Number come determine with it is above-mentioned it is relatively low detection temperature target nucleic acid sequence presence.This example expression, can be by utilizing base
The target with above-mentioned relatively low detection temperature is determined in the presence of signal at a temperature of 2 detections and the difference that is not present
The presence of nucleic acid sequence.
According to an example, in order to calculate above-mentioned difference, the background signal that can will be detected at a temperature of relatively high detection
It is processed into " 0 " or " 1 ".
According to an example, when obtaining negative value in calculating process, negative value is used in a manner of being converted into exhausted angle value
Obtain difference.
An example according to the present invention, the signal for the target nucleic acid sequence with above-mentioned relatively high detection temperature is meter
It calculates parameter (calculation parameter), above-mentioned calculating parameter is for analyzing for above-mentioned relatively low detection temperature
The signal of the target nucleic acid sequence of degree.
For determining the signal detected at a temperature of above-mentioned relatively high detection and in above-mentioned relatively low detection temperature
Under the existing signal of the difference between the signal that detects and the target nucleic acid sequence with above-mentioned relatively high detection temperature can
With mutually different scale (dimensions) or unit (units), and can size or unit having the same.
Term " based on the determination (determined by a difference) of difference " shown in the present specification
Including the generation based on difference/non-generation determination, the value with numerical value difference or based on the determination of range and based on difference
The determination of mapping result.In particular, " based on determination of difference " includes being obtained based on above-mentioned difference for relatively low detection
The determination of the value (for example, CT) of the target nucleic acid of temperature.
" difference (by a is based on the term that the presence of target nucleic acid sequence is used together is determined in the present specification
Difference) " mean by with including the use of the numerical value of difference or its deformation, using signal in the presence/absence of and
The presence of target nucleic acid sequence is directly or indirectly determined using difference or deformation with the mode of threshold value comparison difference.Term " base
In difference " and " utilize difference (by Using a difference) " there is no difference, use in the present specification.
The Mathematical treatment of above-mentioned signal can be carried out by a variety of calculation methods and their deformation.
An example according to the present invention, the Mathematical treatment of the signal for obtaining the difference between above-mentioned signal are to be directed to
The ratio for the signal of the signal detected at a temperature of above-mentioned relatively high detection detected at a temperature of above-mentioned relatively low detection
The calculating of rate.An example according to the present invention, the Mathematical treatment of the signal for obtaining the difference between above-mentioned signal are to be directed to
The signal of the signal detected at a temperature of above-mentioned relatively low detection detected at a temperature of above-mentioned relatively high detection
The calculating of ratio.
Term " ratio (ratio) " used in the present specification means the relationship between 2 numbers.Benefit can be passed through
The presence of the target nucleic acid sequence with above-mentioned relatively low detection temperature is determined with ratio.When in above-mentioned relatively the Supreme People's Procuratorate
It is this existing when the ratio for the signal of the signal detected under testing temperature detected at a temperature of above-mentioned relatively low detection is significant
As becoming the existing indicator (indicator) for the target nucleic acid sequence with relatively low detection temperature for example, taking seriously
To the endpoint intensity of the signal detected at a temperature of above-mentioned relatively high detection in above-mentioned relatively low detection temperature
Under the ratio of the endpoint intensity of signal that detects it is significant when (that is, increase of endpoint intensity), phenomenon indicates to have opposite in this
The presence of the target nucleic acid sequence of lower detection temperature.
Above-mentioned Mathematical treatment can carry out in many ways.
Above-mentioned Mathematical treatment can be carried out using machine.For example, can be anti-by means of detector or Real-Time Polymerase Chain
Equipment processor is answered to carry out Mathematical treatment to signal.It with selecting a property, in particular, can be according to preset algorithm manually
(manually) Mathematical treatment is carried out to above-mentioned signal.
An example according to the present invention, according to for obtain above-mentioned difference close to method, it is obtained above-mentioned in order to analyze
Difference indicates whether the presence with the target nucleic acid sequence of above-mentioned relatively low detection temperature, and threshold value can be used.For example, to consider
From the base comprising the target nucleic acid with above-mentioned relatively high detection temperature and the target nucleic acid with above-mentioned relatively low detection temperature
The mode for the difference that quasi- sample obtains predefines above-mentioned threshold value.In order to which threshold value can also add negative control group, susceptibility
Or used label.
An example according to the present invention, according to for obtain above-mentioned difference close to method, can be by using on obtained
Difference itself is stated to determine the presence of the target nucleic acid sequence with above-mentioned relatively low detection temperature.For example, to above-mentioned opposite
Compared with the signal at a temperature of high detection multiplied by threshold value after, can get the signal at a temperature of the above-mentioned signal that is multiplied and relatively low detection
Between difference.In particular, with consider from comprising with it is above-mentioned it is relatively high detection temperature target nucleic acid and have it is above-mentioned relatively
The mode for the difference that the base sample of the target nucleic acid of low detection temperature obtains predefines above-mentioned threshold value.
An example according to the present invention determines threshold by user's (user) threshold value or automatic (automatically)
Value.
In one example, surely to the above-mentioned relatively the Supreme People's Procuratorate of the target nucleic acid sequence with above-mentioned relatively high detection temperature
When the difference between signal and above-mentioned signal at a temperature of relatively low detection under testing temperature becomes much larger, using above-mentioned
Threshold value reduces detection mistake.
In one example, when the signal provided by the target nucleic acid sequence with above-mentioned relatively high detection temperature is examined at 2
When between testing temperature with undifferentiated or without the slightest difference pattern (or rule), by true in the way of calculating or difference
Surely the existing aspect of the target nucleic acid sequence with above-mentioned relatively low detection temperature, can use in a manner of without additional deformation
The signal detected at a temperature of above-mentioned relatively high detection.
In one example, it is above-mentioned determining in the case that there is pattern (or the rule) for indicating difference in particular range
Target nucleic acid sequence it is existing during, can make the signal at a temperature of above-mentioned relatively high detection in a manner of reflecting above-mentioned difference
Deformation.
Said reference value is the value of the pattern (rule) of the signal intensity at a temperature of the different detections of reflection.
An example according to the present invention, said reference value are reflections for the target nucleic acid sequence with relatively high detection temperature
The value of the pattern (or rule) of signal intensity at a temperature of the different detections of 2 of column.
For example, when for the above-mentioned relatively high detection temperature of the target nucleic acid sequence with above-mentioned relatively high detection temperature
And above-mentioned signal at a temperature of relatively low detection is identical or substantially the same and passes through 2 detection temperature of subtraction of signal
Under signal between difference degree when, at a temperature of with above-mentioned 2 of temperature detections of relatively high detection
The said reference value of signal is " 0 ".As an example again, at a temperature of 2 detections above-mentioned according to the division calculation of above-mentioned signal
When difference between signal, for the said reference of the signal at a temperature of 2 detections with above-mentioned relatively high detection temperature
Value is " 1 ".
On the other hand, the above-mentioned relatively high detection to the target nucleic acid sequence with above-mentioned relatively high detection temperature surely
Above-mentioned signal at a temperature of temperature and relatively low detection is different and according to the difference between above-mentioned 2 signals of subtraction
Cheng Shi, relative to the upper of the signal at a temperature of 2 detections of the target nucleic acid sequence with above-mentioned relatively high detection temperature
Stating a reference value is the positive or negative value other than " 0 ".As another example, when the division calculation according to above-mentioned signal is above-mentioned
When signal at a temperature of 2 detections, relative to 2 of the target nucleic acid sequence detections with above-mentioned relatively high detection temperature
The said reference value of the signal of temperature is to be greater than 1 or the value less than 1 other than " 1 ".
In particular instances, when for the above-mentioned relatively high of the target nucleic acid sequence with above-mentioned relatively high detection temperature
Difference degree category when above-mentioned signal at a temperature of detection temperature and relatively low detection is different, between above-mentioned 2 signals
In particular range.
In particular instances, it can be indicated by a reference value according to the target nucleic acid sequence with above-mentioned relatively high detection temperature
The difference between above-mentioned signal at a temperature of the above-mentioned relatively high detection temperature provided and relatively low detection.In particular instance
In, for according to the above-mentioned relatively high detection temperature and phase that there is the target nucleic acid sequence of above-mentioned relatively high detection temperature to provide
A reference value when different to the above-mentioned signal at a temperature of lower detection compared with a reference value when identical for two signals,
Difference be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%,
7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 12%, 15%, 20% or 30% or more.
In one example, when for the above-mentioned relatively the Supreme People's Procuratorate of the target nucleic acid sequence with above-mentioned relatively high detection temperature
When difference between testing temperature and above-mentioned signal at a temperature of relatively low detection becomes much larger, using for above-mentioned phase
To a reference value of the target nucleic acid sequence compared with the Supreme People's Procuratorate's testing temperature, thus determining the target nucleic acid with above-mentioned relatively low detection temperature
Reduction detection mistake is more advantageous in during the presence of sequence.
In particular instances, there is above-mentioned relatively the Supreme People's Procuratorate for from the signal at a temperature of above-mentioned 2 detections is calculated
The a reference value of the target nucleic acid sequence of testing temperature with for above-mentioned 2 signals it is identical when a reference value compared with, difference 0.5%,
1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%,
8.5%, it in the case where 9%, 9.5%, 10%, 12%, 15%, 20% or 30% or more, is determining with above-mentioned relatively low
Detecting the existing of the target nucleic acid sequence of temperature can be used in the process for the target nucleic acid with above-mentioned relatively high detection temperature
The a reference value of sequence.
According to an example, in order to determine whether to carry out above-mentioned comparison using a reference value, according to signal
Division calculation said reference value.According to an example, be used to determine whether using said reference value method can with for detect
The calculation method for stating a reference value of target nucleic acid sequence is identical or different.
According to an example, in order to according at a temperature of above-mentioned relatively high detection signal and above-mentioned relatively low detection temperature
Under signal between difference come determine with it is above-mentioned it is relatively low detection temperature target nucleic acid sequence presence, and use benchmark
Value.In particular, a reference value is related to having the above-mentioned relatively high detection target nucleic acid sequence of temperature.
An example according to the present invention indicates whether there is above-mentioned relatively low inspection to analyze above-mentioned difference obtained
Said reference value can be used in the presence of the target nucleic acid sequence of testing temperature.
An example according to the present invention, in order to obtain signal at a temperature of above-mentioned relatively high detection and above-mentioned relatively low
Said reference value can be used in the difference between signal at a temperature of detection.For example, can be with above-mentioned relatively high detection temperature
The a reference value of the target nucleic acid sequence of degree is can get above-mentioned as a result, multiplied by or except signal at a temperature of above-mentioned relatively high detection
Multiplied by or difference between the signal and above-mentioned signal at a temperature of relatively low detection that are in can be with as another example
The a reference value of target nucleic acid sequence with above-mentioned relatively high detection temperature is multiplied by or except at a temperature of above-mentioned relatively low detection
Signal, can get the difference between the signal at a temperature of above-mentioned relatively high detection as a result,.
An example according to the present invention uses a reference value for threshold value.An example according to the present invention, a reference value
Using deformation is carried out to above-mentioned value or without deformation in a manner of use as threshold value.In order to by analysis signal between difference come
Determine the presence of target nucleic acid sequence, term " threshold value (threshold) " used in the present specification and " a reference value " can have
There are identical value or identical meaning.
With selecting a property, when in order to obtain signal and the above-mentioned relatively low detection temperature at a temperature of above-mentioned relatively high detection
Under signal between difference and use said reference value when, in order to determine the conspicuousness of above-mentioned difference, that is, above-mentioned in order to determine
Difference indicates whether the presence with the target nucleic acid sequence of relatively low detection temperature, and additional threshold value can be used.
According to an example, in the case where there is the target nucleic acid sequence with relatively high detection temperature, in order to determine tool
There is the presence of the target nucleic acid sequence of relatively low detection temperature, a reference value can be used.
It include detecting for indicating have in the presence of the case where target nucleic acid sequence with above-mentioned relatively high detection temperature
The case where existing significant signal of the target nucleic acid sequence of above-mentioned relatively high detection temperature.
According to an example, in the absence of the target nucleic acid sequence with above-mentioned relatively high detection temperature, in order to determine tool
There is the presence of the target nucleic acid sequence of above-mentioned relatively low detection temperature, optionally uses said reference value.
There is no with it is above-mentioned it is relatively high detection temperature target nucleic acid sequence the case where include only detect have and back
As scape class signal the case where the signal of intensity.
An example according to the present invention has depositing for the above-mentioned relatively low target nucleic acid sequence for detecting temperature to determine
The a reference value obtained as follows is being utilized in the above-mentioned methods, that is, (i) is different from the reaction vessel in above-mentioned steps (a)
In reaction vessel and for detecting the signal-generation mechanism with the detection of target nucleic acid sequence of above-mentioned relatively high detection temperature
Culture has the target nucleic acid sequence of above-mentioned relatively high detection temperature together, and (ii) is in above-mentioned relatively high detection temperature and above-mentioned
After detecting signal at a temperature of relatively low detection, (iii) is detected at a temperature of above-mentioned relatively high detection by finding out
Signal and the signal that is detected at a temperature of above-mentioned relatively low detection between difference obtain said reference value.
According to an example, obtained in above-mentioned (iii) in above-mentioned relatively high detection at a temperature of the signal that detects and
The difference between signal detected at a temperature of above-mentioned relatively low detection be a value, above-mentioned value with without deformation or deform
Mode is used as a reference value.
It, can be by calculating the signal detected at a temperature of above-mentioned relatively high detection and above-mentioned opposite according to an example
The ratio between signal that detects at a temperature of lower detection or subtraction obtain a reference value.An example according to the present invention, can
By calculating examining at a temperature of above-mentioned relatively low detection for the signal detected at a temperature of above-mentioned relatively high detection
The ratio of the signal measured obtains a reference value.An example according to the present invention, by calculating in above-mentioned relatively low inspection
The ratio of the signal of the signal detected under testing temperature detected at a temperature of above-mentioned relatively high detection obtains a reference value.
According to an example, calculation method for the signal difference from sample and for the difference for obtaining a reference value
Calculation method may be the same or different.For example, the signal difference from sample can be carried out according to the subtraction of 2 signals.With selecting a property, from
The signal difference of said sample and difference for obtaining said reference value can removing according to 2 signals for obtaining ratio
Method carries out.
An example according to the present invention, for said reference value signal-generation mechanism can with for detecting above-mentioned target nucleus
Signal-generation mechanism of acid sequence is identical.
It can include ingredient (for example, target nucleic acid sequence, signal-generation mechanism, enzyme or dNTPs) for target nucleic acid sequence
Amount, obtain a reference value under pH of buffer or a variety of reaction conditions in reaction time.An example according to the present invention can react
At the end of provide saturation signal (saturated signal) sufficient reaction condition under obtain a reference value.It is according to the present invention
One example, the difference between signal obtained during calculating benchmark value has particular range, in above-mentioned particular range
Or a reference value is selected referring to above-mentioned particular range.An example according to the present invention, may be selected the maximum of above-mentioned particular range
The maximum value or minimum value of value or minimum value as a reference value or referring to above-mentioned particular range to select a reference value, in particular, examining
Consider standard variation (standard variation), the allowable error range of the said reference value obtained under numerous conditions
(acceptable error ranges), specificity or susceptibility, can be such that a reference value deforms.
An example according to the present invention can include ingredient (if using, just using enzyme or amplimer), pH of buffer
Or a reference value is obtained under identical reaction condition used in the sample of reaction process.An example according to the present invention, it is available
A reference value is obtained with the amplification of signal process of amplification of signal process or the free nucleic acid amplification of nucleic acid amplification.
An example according to the present invention, in order to determine said reference value and with the above-mentioned relatively low target for detecting temperature
The presence of nucleic acid sequence and there are in the case where significant difference between the difference that obtains, there is above-mentioned relatively low detection temperature
Target nucleic acid sequence be confirmed as existing.Said reference value, which is represented by, has above-mentioned relatively low detection temperature in order to determine
Target nucleic acid sequence presence and the value (for example, ratio of the endpoint value of signal strength) of the identical type of difference that obtains.
In particular case, when the endpoint value relative to the signal strength detected at a temperature of above-mentioned relatively low detection
The ratio of the endpoint value of the signal strength detected at a temperature of above-mentioned relatively high detection is 1.8 and said reference value is 1.1
When, the presence in order to determine the target nucleic acid sequence with said reference value and above-mentioned relatively low detection temperature can be determined to be in
And there is significant difference between the difference obtained.This indicates there is the target nucleic acid sequence with above-mentioned relatively low detection temperature.
According to an example, when for determining the existing difference with the target nucleic acid sequence of above-mentioned relatively low detection temperature
It is identical as said reference value or be greater than said reference value when, be determined as exist with it is above-mentioned it is relatively low detection temperature target nucleic acid
Sequence.
According to an example, when for determining the existing difference with the target nucleic acid sequence of above-mentioned relatively low detection temperature
When with said reference value identical or less than said reference value, it is determined as the target nucleic acid for having with above-mentioned relatively low detection temperature
Sequence.
With selecting a property, in order to calculate the signal detected at a temperature of above-mentioned relatively high detection and above-mentioned relatively low
Said reference value can be used in the difference between signal detected at a temperature of detection.For example, use can be calculated by following mode
In the existing difference for determining the target nucleic acid sequence with relatively low detection temperature, that is, in above-mentioned relatively high detection temperature
Under after a reference value of the signal (for example, RFU) that detects multiplied by the target nucleic acid sequence with above-mentioned relatively high detection temperature
(or divided by later), subtracted from above-mentioned multiplication (or division) result detected at a temperature of relatively low detection signal (for example,
RFU).In the case where difference is greater than (or being less than) " 0 " or predetermined value, it may be determined that exist with above-mentioned relatively low
Detect the target nucleic acid sequence of temperature.
As another example, can be calculated by following mode for determining the target nucleic acid sequence with relatively low detection temperature
The existing difference of column, that is, the signal (for example, RFU) detected at a temperature of above-mentioned relatively low detection is multiplied by with above-mentioned
After (or divided by later) a reference value of the target nucleic acid sequence of relatively high detection temperature, subtract from above-mentioned multiplication (or division) result
Remove the signal (for example, RFU) detected at a temperature of relatively high detection.It is greater than in difference (or being less than) " 0 " or predetermined
In the case where value, it may be determined that there is the target nucleic acid sequence with above-mentioned relatively low detection temperature.
According to an example, predetermined value can play threshold function.
According to an example, when detect for the target nucleic acid sequence with above-mentioned relatively high detection temperature signal or
Signal at a temperature of above-mentioned relatively high detection is obtained by mathematical processes and above-mentioned at a temperature of relatively low detection
When difference between signal, used above-mentioned to determine the presence of the target nucleic acid sequence with above-mentioned relatively low detection temperature
A reference value.
According to an example, when generating signal with real-time mode relevant to the target amplification based on polymerase chain reaction,
The Mathematical treatment of above-mentioned signal includes detecting at a temperature of above-mentioned relatively low detection for during each amplification cycles
Signal strength the signal strength detected at a temperature of above-mentioned relatively high detection ratio calculation.Above-mentioned calculated result needle
It draws to circulation, and for determining the presence with the target nucleic acid sequence of above-mentioned relatively low detection temperature.
According to an example, when generating signal with real-time mode relevant to the target amplification based on polymerase chain reaction,
CtValue is the signal detected for target.
The signal detected at a temperature of above-mentioned relatively high detection temperature and above-mentioned relatively low detection can be utilized true
Surely the C of the target nucleic acid sequence with above-mentioned relatively low detection temperaturetValue, it is available to be illustrated in the following example: firstly, to needs
The sample of analysis carries out real-time polymerase chain reaction, is obtaining in above-mentioned relatively high detection temperature and above-mentioned relatively low inspection
After the signal detected under testing temperature, the amplification curve of above-mentioned 2 detections temperature is obtained.
(a) in detection process at a temperature of above-mentioned relatively high detection, when with above-mentioned relatively high detection temperature
The C of target nucleic acid sequencetIn the absence of value, it may be determined that for there is no the target nucleic acid sequences with above-mentioned relatively high detection temperature.
Then, the target nucleic acid with above-mentioned relatively low detection temperature is calculated from the amplification curve obtained at a temperature of relatively low detection
The C of sequencetValue.When the target nucleic acid sequence with above-mentioned relatively low detection temperature is also not present, then with above-mentioned relatively low
Detect the C of the target nucleic acid sequence of temperaturetValue is also not present.
(b) in detection process at a temperature of above-mentioned relatively high detection, when with above-mentioned relatively high detection temperature
The C of target nucleic acid sequencetIn the presence of value, C is being indicatedtIt calculates to be directed in the circulation of value and be obtained at a temperature of above-mentioned relatively high detection
RFU value the RFU value obtained at a temperature of above-mentioned relatively low detection ratio.Also, it also calculates and notationally states CtValue
Circulation after circulation in the ratio that obtains.(i) the ratio of all RFU values be less than a reference value (for example, as described above,
The value obtained merely with the target nucleic acid sequence with above-mentioned relatively high detection temperature) in the case where, it is determined as that tool is not present
There is the target nucleic acid sequence of above-mentioned relatively low detection temperature.Therefore, without the target nucleic acid sequence with above-mentioned relatively low detection temperature
The C of columntValue.(ii) in the case where the ratio of all RFU values is greater than said reference value, at a temperature of above-mentioned relatively low detection
The calculated C of the amplification curve of acquisitiontValue is confirmed as the C with the target nucleic acid sequence of above-mentioned relatively low detection temperaturetValue.
(iii) as the above-mentioned C of expressiontThe ratio of RFU value in the cyclic process of value is less than the RFU after said reference value and particular cycle
When the ratio of value is greater than said reference value, above-mentioned particular cycle is confirmed as the target nucleic acid with above-mentioned relatively low detection temperature
The C of sequencetValue.
Under the ratio and the identical situation of said reference value being computed, above-mentioned determination can be arbitrarily realized.For example, above-mentioned
Example in, carry out above-mentioned determination in a manner of considering whether above-mentioned ratio is less than or greater than a reference value.
With selecting a property, the C of target nucleic acid sequence of the calculating with above-mentioned relatively low detection temperature that can be followingtValue, that is, In
In each circulation, calculate for the RFU value obtained at a temperature of above-mentioned relatively high detection in above-mentioned relatively low detection temperature
After the ratio of the lower RFU value obtained of degree, threshold value is considered to calculate CtValue.
With selecting a property, the C of target nucleic acid sequence of the calculating with above-mentioned relatively low detection temperature that can be followingtValue, that is, benefit
Deform the RFU value obtained at a temperature of the above-mentioned relatively high detection of each circulation with a reference value of each circulation, right
Each cycle calculations for above-mentioned deformation RFU value the RFU value obtained at a temperature of above-mentioned relatively low detection ratio it
Afterwards, C is calculatedtValue.
An example according to the present invention includes using the step of signal detected at a temperature of above-mentioned relatively high detection
Obtain the existing limit value (qualifying for determining the target nucleic acid sequence with above-mentioned relatively high detection temperature
Value) the step of includes obtaining for determining the target nucleus with above-mentioned relatively low detection temperature using the step of above-mentioned difference
Acid sequence there are the step of.
An example according to the present invention is used to determine with above-mentioned relatively low using the step of above-mentioned difference including obtaining
The step of detecting the existing limit value of the target nucleic acid sequence of temperature, when (i) is detected at a temperature of above-mentioned relatively high detection
Signal and the signal progress Mathematical treatment detected at a temperature of above-mentioned relatively low detection or (ii) out is above-mentioned opposite
When compared with signal is not detected at a temperature of high detection, it is contemplated that do not detect signal at a temperature of above-mentioned relatively high detection, pass through
Above-mentioned limit value is obtained using the signal detected at a temperature of above-mentioned relatively low detection.
In order to obtain the value of deformation, can also Mathematical treatment be carried out to above-mentioned limit value.Above-mentioned limit value is for determining sample
The presence of interior 2 target nucleic acid sequences.
According to an example, said one reaction vessel further includes at least one attachment device (additional set), above-mentioned
At least one attachment device (additional set) is respectively included for detecting the target other than above-mentioned 2 target nucleic acid sequences
2 additional signal-generation mechanisms of nucleic acid sequence, are mutually distinguishable in said vesse by 2 each dresses of signal-generation mechanism
The above-mentioned signal for setting generation detects above-mentioned signal by different types of detector respectively.For example, when marking above-mentioned step with FAM
Suddenly two signal-generation mechanisms in (a) and when marking 2 additional signal-generation mechanisms with Quasar 570, in above-mentioned appearance
The signal generated in device by the signal-generation mechanism marked by FAM- is produced with the signal-generation mechanism marked by Quasar 570-
Raw signal is mutually distinguishable, it is therefore desirable to for detecting 2 seed types of 2 different releasing light (emission lights)
Detector.
An example according to the present invention, above-mentioned 2 target nucleic acid sequences include nucleotide diversity (nucleotide
Variation), one in above-mentioned 2 target nucleic acid sequences includes a type of nucleotide diversity, another includes another kind of
The nucleotide diversity of type.
Term " nucleotide diversity (nucleotide variation) " used in the present specification means even
All single or multiple nucleotide subsitutions in continuous deoxyribonucleic acid fragment in the DNA sequence of specific position,
Missing or insertion.This continuous deoxyribonucleic acid fragment includes other any positions of a gene or a chromosome.
This nucleotide diversity can be mutation (mutant) or polymorphic allele variation (polymorphic allele
variations).For example, the nucleotide diversity detected in the present invention includes single nucleotide polymorphism (single
Nucleotide polymorphism, SNP), mutation (mutation), missing, insertion, displacement and displacement.Nucleotide diversity
Example include various variations in human genome (for example, methylenetetrahydrofolate reductase
The variation of (methylenetetrahydrofolate reductase, MTHFR) gene), the drug-resistant of pathogen it is relevant
Variation and tumour formation-Induced variation.Term " nucleotide diversity " used in the present specification includes nucleic acid sequence
All variations of specific position.That is, term " nucleotide diversity " includes the wild type of the specific position of nucleic acid sequence and its owns
Saltant type.
An example according to the present invention is single nucleotide polymorphism by the bright nucleotide diversity detected of this law
(single nucleotide polymorphism；Nucleotide polymorphisms).
An example according to the present invention, a gene in above-mentioned nucleotide polymorphisms allele have by corresponding
The above-mentioned relatively high detection temperature that above-mentioned signal-generation mechanism determines, another gene have by corresponding above-mentioned signal-
The above-mentioned relatively low detection temperature that generation mechanism determines.
Advantages of the present invention can be further emphasized in nucleotide polymorphisms detection.
Detection temperature for wild-type allele (allele) is relatively high detection temperature, for mutation equipotential base
The detection temperature of cause is relatively low detection temperature, in the case where sample is no mutant homozygote (homozygous), above-mentioned
Signal cannot be detected at a temperature of relatively high detection, and detects signal at a temperature of above-mentioned relatively high detection.Above-mentioned examination
Sample is determined as not including wild-type allele, when including mutant allele.On the other hand, it is tried for no mutant homozygote
Sample, even if in the case where generating the signal of mistake at a temperature of above-mentioned relatively high detection, it can also be by calculating for determining tool
There is the nucleotide polymorphisms allele of above-mentioned relatively low detection temperature is existing to examine at a temperature of above-mentioned relatively high detection
The result of difference between the signal measured and the signal detected at a temperature of above-mentioned relatively low detection confirms relatively
Whether the signal detected at a temperature of high detection is false positive signal.Above-mentioned reason is the heterozygote for nucleotide polymorphisms
It (heterozygote) include wild-type allele and mutation allele with the ratio of 1:1.
II. different detection temperature analysis nucleotide polymorphisms genotype (SNP Genotyping) are utilized
In a further embodiment of the invention, the present invention, which provides, utilizes different detection temperature analysis sample nucleic acid sequences
Nucleotide polymorphisms genotype method, it is above-mentioned utilize it is different detection temperature analysis sample nucleic acid sequences nucleotide polymorphisms
Property genotype method include: step (a), in 1 reaction vessel and for detecting the letter of nucleotide polymorphisms allele
Number-generation mechanism cultivates the sample comprising the nucleic acid sequence containing above-mentioned mononucleotide polymorphism site together, and using single
The detector of type detects generated signal, detects each above-mentioned nucleotide polymorphisms by corresponding signal-generation mechanism
Allele, one in above-mentioned nucleotide polymorphisms allele has the phase determined by corresponding signal-generation mechanism
To compared with the Supreme People's Procuratorate's testing temperature, another has the relatively low detection temperature determined by corresponding signal-generation mechanism, above-mentioned phase
To compared with the Supreme People's Procuratorate's testing temperature be can generate for it is above-mentioned it is relatively high detection temperature nucleotide polymorphisms allele
The temperature of signal, above-mentioned relatively low detection temperature are that can generate for the nucleotide with above-mentioned relatively low detection temperature
The signal and needle set of polymorphic allele have the letter of the nucleotide polymorphisms allele of above-mentioned relatively high detection temperature
Number temperature, the signal generated by above-mentioned signal-generation mechanism is not distinguished by the detector of above-mentioned single type, above-mentioned opposite
Compared with carrying out above-mentioned detection at a temperature of the Supreme People's Procuratorate's testing temperature and above-mentioned relatively low detection；And step (b), according to above-mentioned steps
(a) it the signal detected at a temperature of above-mentioned relatively high detection in and is detected at a temperature of above-mentioned relatively low detection
Signal between difference determine nucleotide polymorphisms genotype.
The present invention is based on the principles in above-mentioned first embodiment of the invention, therefore, in order to avoid leading to this explanation
The excessive repetition of the complexity of book, omits the common content between them.In order to illustrate present embodiment, and it is related to for upper
In the case where the explanation for stating first embodiment, the step of being present embodiment with needing to pay attention to (b) and first embodiment
The step of (b) it is a part of complementary different.Therefore, general technical staff of the technical field of the invention should be understood that for first
A part explanation of a embodiment can be directly applied for the explanation of (b) the step of for present embodiment, by its of deformation
He illustrates the explanation for being applicable to (b) the step of for present embodiment.
Step (a): with the culture of signal-generation mechanism and signal detection
Firstly, signal-generation mechanism one in a reaction vessel and for detecting nucleotide polymorphisms allele
After sample of the culture comprising the nucleic acid sequence containing above-mentioned mononucleotide polymorphism site, examined using the detector of single type
Signal caused by surveying.The signal generated by above-mentioned signal-generation mechanism is not distinguished by the detector of single type.
Nucleic acid sequence comprising above-mentioned nucleotide polymorphic site may include the chromosome pair of the mankind.
One in above-mentioned nucleotide polymorphisms allele has by corresponding above-mentioned signal-generation mechanism determination
Relatively high detection temperature, another has the relatively low detection temperature determined by corresponding above-mentioned signal-generation mechanism,
Above-mentioned relatively high detection temperature is that can generate for the nucleotide polymorphisms equipotential with above-mentioned relatively high detection temperature
The temperature of the signal of gene, above-mentioned relatively low detection temperature are that can generate for above-mentioned relatively low detection temperature
The signal and needle set of nucleotide polymorphisms allele have the nucleotide polymorphisms equipotential base of above-mentioned relatively high detection temperature
The temperature of the signal of cause,
An example according to the present invention carries out above-mentioned steps (a) during with the amplification of signal of nucleic acid amplification.
An example according to the present invention carries out above-mentioned steps (a) during the amplification of signal of free nucleic acid amplification.
Step (b): the determination of nucleotide polymorphisms genotype
After detecting signal, according to the letter detected at a temperature of above-mentioned relatively high detection in above-mentioned steps (a)
Number and the signal that is detected at a temperature of above-mentioned relatively low detection between difference determine nucleotide polymorphisms genotype.
In the present invention, even if without for the nucleotide polymorphisms allele with above-mentioned relatively high detection temperature
It determines, only by the signal detected at a temperature of above-mentioned relatively high detection and at a temperature of above-mentioned relatively low detection
The difference between signal detected carrys out analysis of nucleotide Genetic polymorphism type.
According to an example, by the signal detected at a temperature of above-mentioned relatively high detection and above-mentioned relatively low
The signal detected at a temperature of detection carries out Mathematical treatment, to obtain above-mentioned difference.
According to an example, by calculate the signal detected at a temperature of above-mentioned relatively high detection and it is above-mentioned relatively
The difference between signal that detects at a temperature of low detection obtains above-mentioned difference.
According to an example, the background detected at a temperature of above-mentioned relatively high detection is used in order to calculate above-mentioned difference
Signal.
According to an example, in order to calculate above-mentioned difference, the background signal that can will be detected at a temperature of relatively high detection
It is processed into " 0 " or " 1 ".
According to an example, in the case where negative value is obtained during being started, in order to obtain difference, negative value can be turned
Be changed to absolute value come using.
An example according to the present invention, can be not determine the nucleotide polymorphisms with above-mentioned relatively high detection temperature
The existing mode of allele carries out the above-mentioned steps (b) for determining nucleotide polymorphisms genotype.Using opposite
Come compared with the difference between the signal detected at a temperature of high detection and the signal detected at a temperature of above-mentioned relatively low detection
Carry out nucleotide polymorphisms genotyping.
Do not need determine have it is above-mentioned it is relatively high detect temperature nucleotide polymorphisms allele there are the reasons why
It is, there are 3 nucleotide polymorphisms genotype, and include open country with the ratio of 1:1 for the heterozygote of nucleotide polymorphisms
Raw type allele and mutated-genotype.By combination the principle of the present invention and above-mentioned reason, even if not determining that there is above-mentioned phase
It can also analysis of nucleotide Genetic polymorphism type to the presence of the nucleotide polymorphisms allele compared with the Supreme People's Procuratorate's testing temperature.
An example according to the present invention includes the nucleotide polymorphisms allele with above-mentioned relatively high detection temperature
Homozygote (homozygote) sample indicate particular range in difference (for example, ratio), heterozygote sample indicate other spy
Determine the difference (for example, ratio) in range, includes the pure of the above-mentioned relatively low nucleotide polymorphisms allele for detecting temperature
Zygote indicates the difference (for example, ratio) in another particular range.
An example according to the present invention, for each nucleotide polymorphisms genotype particular range can be directed to each core
The a reference value of nucleotide polymorphism genotype is related.
An example according to the present invention, it is above-mentioned to utilize different detection temperature point in order to determine nucleotide polymorphisms genotype
The method for analysing the nucleotide polymorphisms genotype of sample nucleic acid sequence is utilized for by with above-mentioned relatively high detection temperature
Nucleotide polymorphisms allele formed homozygote, by nucleotide polymorphisms etc. with above-mentioned relatively low detection temperature
It is at least one kind of in a reference value of homozygote and heterozygote that position gene is formed.
An example according to the present invention, it is above-mentioned to utilize different detection temperature point in order to determine nucleotide polymorphisms genotype
The method for analysing the nucleotide polymorphisms genotype of sample nucleic acid sequence uses all above-mentioned 3 a reference values.It is according to the present invention
One example, in order to determine nucleotide polymorphisms genotype, the above-mentioned core using different detection temperature analysis sample nucleic acid sequences
The method of nucleotide polymorphism genotype is utilized by the nucleotide polymorphisms allele shape with above-mentioned relatively high detection temperature
At homozygote and heterozygote at least two a reference value.
It is above-mentioned using in different detection temperature analysis samples in order to determine nucleotide polymorphisms genotype according to an example
The a reference value that obtains as follows is utilized in the method for the nucleotide polymorphisms genotype of nucleic acid sequence, that is, (i) with above-mentioned steps
(a) in other different reaction vessels of reaction vessel in and for detect with the above-mentioned relatively high nucleotide for detecting temperature
Signal-generation mechanism of polymorphic allele is cultivated together by the nucleotide polymorphisms with above-mentioned relatively high detection temperature
The homozygote that allele is formed, (ii) is detected at a temperature of above-mentioned relatively high detection temperature and above-mentioned relatively low detection
After signal, (iii) is by finding out the signal detected at a temperature of above-mentioned relatively high detection and in above-mentioned relatively low inspection
The difference between signal that detects under testing temperature obtains said reference value.
It is above-mentioned using in different detection temperature analysis samples in order to determine nucleotide polymorphisms genotype according to an example
The a reference value that obtains as follows is utilized in the method for the nucleotide polymorphisms genotype of nucleic acid sequence, that is, (i) with above-mentioned steps
(a) heterozygote, above-mentioned heterozygote are cultivated together with corresponding signal-generation mechanism in the different reaction vessel of reaction vessel in
By the nucleotide polymorphisms allele with above-mentioned relatively high detection temperature and with above-mentioned relatively low detection temperature
Nucleotide polymorphisms allele is formed, and (ii) is equal at a temperature of above-mentioned relatively high detection temperature and above-mentioned relatively low detection
Detect signal after, (iii) by find out the signal detected at a temperature of above-mentioned relatively high detection and it is above-mentioned relatively
The difference between signal that detects at a temperature of low detection obtains a reference value.
It is above-mentioned using in different detection temperature analysis samples in order to determine nucleotide polymorphisms genotype according to an example
The a reference value that obtains as follows is utilized in the method for the nucleotide polymorphisms genotype of nucleic acid sequence, that is, (i) with above-mentioned steps
(a) in the different reaction vessel of reaction vessel in and for detect with the above-mentioned relatively low nucleotide polymorphism for detecting temperature
Signal-generation mechanism of property allele cultivates homozygote together, and above-mentioned homozygote is by with above-mentioned relatively low detection temperature
Nucleotide polymorphisms allele formed, (ii) is at a temperature of above-mentioned relatively high detection temperature and above-mentioned relatively low detection
After detecting signal, (iii) is by finding out the signal detected at a temperature of above-mentioned relatively high detection and above-mentioned opposite
The difference between signal that detects at a temperature of lower detection obtains a reference value.
According to an example, in order to analyze the nucleotide polymorphisms genotype of sample, calculate in above-mentioned relatively high detection temperature
Difference between the signal detected under degree and the signal detected at a temperature of above-mentioned relatively low detection, and with each nucleosides
The a reference value of sour Genetic polymorphism type is compared.
An example according to the present invention can be provided at the end of reaction and be obtained under the sufficient reaction condition of saturation signal
State a reference value.For example, being directed to obtain by there is the nucleotide polymorphisms allele with above-mentioned relatively high detection temperature
And a reference value of the heterozygote of the nucleotide polymorphisms allele formation with above-mentioned relatively low detection temperature, it selects and each
The identical reaction condition of content of a nucleotide polymorphisms allele is more for each nucleotide to provide at the end of reaction
The saturation signal of state property allele.An example according to the present invention, the above-mentioned letter obtained in said reference value calculating process
Difference between number has particular range, and said reference value is selected in above-mentioned particular range or is come referring to above-mentioned particular range
Select said reference value.
Detection temperature for above-mentioned wild-type allele is above-mentioned relatively high detection temperature, for above-mentioned mutation etc.
The detection temperature of position gene is above-mentioned relatively low detection temperature, is detected at a temperature of above-mentioned relatively high detection when by calculating
It is above-mentioned wild when ratio between signal out and the signal detected at a temperature of above-mentioned relatively low detection is to obtain difference
Type homozygote sample indicates the ratio in particular range, and heterozygote sample indicates the ratio in other particular ranges.
For example, wild-type homozygote sample indicates about 1.0 ratio, indicated for the heterozygote sample of nucleotide polymorphisms
About 2.0 ratio.
When sample be the mutant homozygous period of the day from 11 p.m. to 1 a.m, in order to calculate the signal detected at a temperature of above-mentioned relatively high detection and
The ratio between signal detected at a temperature of above-mentioned relatively low detection may be used at a temperature of above-mentioned relatively high detection and examines
The background signal measured.In this case, calculated ratio can indicate to be far longer than 2.0 value, the core of this representing sample
Nucleotide polymorphism genotype is no mutant homozygote.With selecting a property, calculated ratio can indicate to belong to according to heterozygous mutation table
The value of the ratio (for example, about 9.0) of the particular range shown.
In particular, no mutant homozygote sample is directed to, even if the case where generating the signal of mistake at a temperature of relatively high detection
Under, above-mentioned ratio also illustrate that be far longer than 2.0 value, the nucleotide polymorphisms genotype in this representing sample is mutant homozygous
Son.
It therefore, can be merely in above-mentioned relatively the Supreme People's Procuratorate in the present invention for nucleotide polymorphisms genotype
The mode of difference between the signal detected under testing temperature and the signal detected at a temperature of above-mentioned relatively low detection is true
Determine nucleotide polymorphisms genotype.
According to an example, above-mentioned difference provide by the signal detected at a temperature of above-mentioned relatively high detection and
The limit value that the signal detected at a temperature of above-mentioned relatively low detection carries out Mathematical treatment to obtain.
According to an example, obtained using the standard sample comprising wild-type homozygote, no mutant homozygote or heterozygote
A reference value is stated, said reference value is for analyzing the difference obtained from experimental sample.
III. at least three target nucleic acid sequence in different detection temperature detection samples is utilized
In another embodiment of the present invention, the present invention, which provides, utilizes at least three in different detection temperature detection samples
The method of target nucleic acid sequence, the above-mentioned method using at least three target nucleic acid sequence in different detection temperature detection samples includes: step
Suddenly (a), at least three signal-generation mechanism one in 1 reaction vessel and for detecting above-mentioned at least three target nucleic acid sequence
Generated signal is detected with culture said sample, and using the detector of single type, by corresponding signal-generation mechanism
Above-mentioned at least three target nucleic acid sequence is detected respectively, and above-mentioned at least three target nucleic acid sequence is respectively provided with by corresponding above-mentioned letter
Number-the determining different detection temperature of generation mechanism, above-mentioned detection temperature is that can not only generate the target with above-mentioned detection temperature
Nucleic acid sequence, additionally it is possible to generate the temperature of the signal for the target nucleic acid sequence with the detection temperature higher than above-mentioned detection temperature
Degree, the signal generated by above-mentioned signal-generation mechanism are not distinguished by the detector of above-mentioned single type, in each different detection temperature
Degree is lower to carry out above-mentioned detection；And step (b), at least three target nucleic acid is determined according to the signal detected in above-mentioned steps (a)
The presence of sequence, as determining particular detection temperature (certain detection having in above-mentioned at least three target nucleic acid sequence
Temperature target nucleic acid sequence) there are when, according to be higher than above-mentioned particular detection temperature 1 or more detection temperature
Difference between the signal detected under degree and the signal detected at a temperature of above-mentioned particular detection determines there is above-mentioned spy
The presence of the target nucleic acid sequence of regular inspection testing temperature, above-mentioned particular detection temperature is opposite Supreme Procuratorate's thermometric in above-mentioned detection temperature
Spend relatively highest detection temperature) when, it is detected according at a temperature of above-mentioned particular detection
Signal determine the presence of above-mentioned target nucleic acid sequence.
The present invention is based on the principles of the first embodiment of aforementioned present invention, therefore, in order to avoid leading to this specification
The excessive repetition of complexity, omits the common content between them.
Step (a): with the culture of signal-generation mechanism and signal detection
Firstly, signal-generation mechanism one in a reaction vessel and for detecting above-mentioned at least three target nucleic acid sequence
After cultivating the sample to be analyzed together, generated signal is detected using the detector of single type.By above-mentioned signal-generation
The signal that mechanism generates is not distinguished by the detector of single type.
In a reaction vessel, according to the present invention the quantity of detected above-mentioned target nucleic acid sequence include 3,4,5,6,7,
8,9 and 10 or more target nucleic acid sequences, but not limit and this.
Each above-mentioned at least three target nucleic acid sequence is detected by corresponding signal-generation mechanism.Each above-mentioned at least three
Target nucleic acid sequence has the different detection temperature determined by corresponding above-mentioned signal-generation mechanism.
According to an example, for the above-mentioned detection temperature that target nucleic acid sequence point is matched mutually have at least 2 DEG C, 3 DEG C, 4 DEG C,
5 DEG C, 7 DEG C, 8 DEG C, 9 DEG C, 10 DEG C, 11 DEG C, 12 DEG C, 15 DEG C or 20 DEG C of difference.
One in above-mentioned target nucleic acid sequence has opposite highest detection temperature.In order to detect with above-mentioned opposite Supreme Procuratorate
The target nucleic acid sequence of testing temperature, using signal-generation mechanism of signal can be provided at a temperature of above-mentioned opposite highest detection.
Detection temperature is not only that the signal for the target nucleic acid sequence with above-mentioned detection temperature can be generated, but also is
It can produce the temperature of the signal for the target nucleic acid sequence with the detection temperature higher than above-mentioned detection temperature.In each different inspections
Above-mentioned detection is carried out under testing temperature.
According to an example, above-mentioned steps (a) is carried out during with the amplification of signal of nucleic acid amplification.
According to an example, above-mentioned steps (a) is carried out during the amplification of signal of free nucleic acid amplification.
According to an example, at least one in signal-generation mechanism is to generate signal in a manner of being formed dependent on dimer
Signal-generation mechanism.
According to an example, signal-generation mechanism for above-mentioned each target nucleic acid sequence is that the formation based on dimer produces
Signal-generation mechanism of raw signal.
According to an example, based on the detection oligonucleotides with target nucleic acid sequence and with above-mentioned target nucleic acid sequence specific hybrid
Between dimer formed and generate above-mentioned signal.According to an example, based on dependent on miscellaneous with above-mentioned target nucleic acid sequence specificity
The dimer that the mode of the cutting of the mediation oligonucleotides of friendship is formed generates above-mentioned signal.
An example according to the present invention, signal-generation mechanism for above-mentioned each target nucleic acid sequence are based on to rely on
In the signal-generation for the dimer that the mode of the cutting of the mediation oligonucleotides with above-mentioned target nucleic acid sequence specific hybrid is formed
Mechanism.
According to an example, at least one in signal-generation mechanism is in a manner of the cutting dependent on detection oligonucleotides
Generate signal-generation mechanism of signal.
According to an example, after above-mentioned detection oligonucleotides hybridizes with target nucleic acid sequence, based on above-mentioned detection oligonucleotides
Cutting generates above-mentioned signal.According to an example, depended on by detecting the cutting of oligonucleotides special with above-mentioned target nucleic acid sequence
The mode of the cutting of the mediation oligonucleotides of specific hybridization generates above-mentioned signal.
According to an example, the signal generating method in a manner of dependent on the cutting of above-mentioned detection oligonucleotides is used to have
State the target nucleic acid sequence of the above-mentioned opposite highest detection temperature at least three target nucleic acid sequence.
An example according to the present invention, the so signal-to the target nucleic acid sequence with above-mentioned opposite highest detection temperature
Generation mechanism is signal-generation mechanism of the cutting based on detection oligonucleotides, for the signal-of remaining above-mentioned target nucleic acid sequence
Generation mechanism is signal-generation mechanism of the formation based on dimer.
An example according to the present invention, for signal-production of the target nucleic acid sequence with above-mentioned opposite highest detection temperature
Life structure is signal-generation mechanism of the cutting based on detection oligonucleotides, for signal-production of remaining above-mentioned target nucleic acid sequence
Life structure is based on being formed in a manner of dependent on the cutting of the mediation oligonucleotides of above-mentioned target nucleic acid sequence specific hybrid
Signal-generation mechanism of dimer.
An example according to the present invention releases segment, above-mentioned fragment mediates two in the cutting of above-mentioned mediation oligonucleotides
The formation of polymers or the cutting for detecting oligonucleotides that the extension based on above-mentioned segment is mediated in capturing oligonucleotides.
An example according to the present invention, above-mentioned at least three signal-generation mechanism includes identical label, from above-mentioned label
Signal not by above-mentioned single type detector distinguish.
An example according to the present invention is generated in a manner of the formation dependent on dimer in above-mentioned signal-generation mechanism
In the case where signal, the Tm value based on above-mentioned dimer selects above-mentioned detection temperature.
An example according to the present invention, in above-mentioned signal-generation mechanism with the side of the cutting dependent on detection oligonucleotides
In the case that formula generates signal, above-mentioned detection temperature is arbitrarily selected.An example according to the present invention is based on above-mentioned detection few nucleosides
Signal-generation mechanism of the cutting of acid can provide opposite highest detection temperature.
According to an example, it is contemplated that the temperature range that can generate signal by above-mentioned signal-generation mechanism is predefined and is directed to
The detection temperature of target nucleic acid sequence.
According to an example, it is contemplated that signal can be generated by the signal for detecting each target nucleic acid sequence-generation mechanism
Temperature range, predefine for above-mentioned each target nucleic acid sequence detection temperature.In view of not being overlapped in detection temperature
Detection temperature range and overlapping detection temperature orientation, above-mentioned detection temperature can be predefined.
Detection at a temperature of above-mentioned particular detection is applied not only to detection for the target nucleic acid sequence with above-mentioned detection temperature
Signal, but also for detect for be higher than above-mentioned detection temperature detection temperature target nucleic acid sequence signal.On
The detection at a temperature of opposite highest detection is stated for detect for the above-mentioned target nucleic acid sequence of highest detection temperature relatively
Signal.
For example, including three target sequences in above-mentioned target nucleic acid sequence, divide respectively in 72 DEG C, 60 DEG C and 50 DEG C of detection temperature
In the case where assigned in above three target sequence, the detection at a temperature of above-mentioned 50 DEG C not only includes for 50 DEG C of detection temperature
Target nucleic acid sequence signal detection, but also including for be respectively provided with 70 DEG C and 60 DEG C detection temperature target nucleic acid sequences
Signal detection.
One of the features of the present invention is to indicate above-mentioned at least three target nucleic acid sequence by detecting at a temperature of different detections
Existing signal, Lai Yiqu determine the presence of above-mentioned at least three target nucleic acid sequence otherwise.
Step (b): the presence of at least three target nucleic acid sequence is determined
After the detection for completing signal, at least three target nucleic acid sequence is determined according to the signal detected in above-mentioned steps (a)
The presence of column.
According to an example, in order to confirm whether the signal detected at a temperature of particular detection includes according to above-mentioned spy
The signal that the target nucleic acid sequence of regular inspection testing temperature provides, and utilize and detected at a temperature of being higher than the detection of above-mentioned particular detection temperature
Signal out, and target nucleic acid sequence is determined in a manner of analyzing the signal detected at a temperature of above-mentioned particular detection
In the presence of.
In the presence of determining the target nucleic acid sequence with particular detection temperature at least three target nucleic acid sequence,
According to the signal detected at a temperature of 1 or more the detection higher than above-mentioned particular detection temperature and in above-mentioned particular detection temperature
Difference between the signal detected under degree determines the presence with the target nucleic acid sequence of above-mentioned particular detection temperature.In above-mentioned inspection
In testing temperature, in the case that above-mentioned particular detection temperature is opposite highest detection temperature, according at a temperature of above-mentioned particular detection
The signal detected determines the presence of above-mentioned target nucleic acid sequence.
According to an example, above-mentioned difference includes in 1 or more detection temperature for being higher than above-mentioned particular detection temperature
Under the signal detected and the signal detected at a temperature of above-mentioned particular detection carry out the difference that the mode of Mathematical treatment obtains.
It is examined according to an example in the case where not detecting signal at a temperature of the detection for being higher than above-mentioned particular detection temperature
Consider and do not detect signal at a temperature of being higher than the detection of above-mentioned particular detection temperature, is examined according at a temperature of above-mentioned particular detection
The signal measured determines the presence with the target nucleic acid sequence of above-mentioned particular detection temperature.This example can be by using because of signal
Presence and difference caused by being not present determine the presence of the target nucleic acid sequence with above-mentioned particular detection temperature.
For example, it is assumed that target nucleic acid sequence includes 4 target sequences, and 72 DEG C, 60 DEG C, 50 DEG C and 40 DEG C of detection temperature is divided
It is not allocated in above-mentioned target sequence, then in above-mentioned steps (a), detects signal at a temperature of 60 DEG C, 50 DEG C and 40 DEG C of detection.
In the presence of determining the target nucleic acid sequence with above-mentioned 40 DEG C detection temperature, using higher than above-mentioned particular detection temperature
It spends the signal detected at 1 or more the detection temperature (60 DEG C or 50 DEG C) of (40 DEG C) and is examined at a temperature of above-mentioned particular detection
The difference between signal measured, to determine the target nucleic acid sequence with above-mentioned particular detection temperature (40 DEG C).More clearly, it is
Determine the presence with the target nucleic acid sequences of 40 DEG C of detection temperature, the signal that is detected at a temperature of maying be used at 60 DEG C and
The difference between signal that is detected at a temperature of 40 DEG C, 50 DEG C at a temperature of the signal that detects and 40 DEG C at a temperature of
The difference or above-mentioned 2 difference between signal detected.
The target nucleic acid sequence with other detections temperature (that is, 60 DEG C and 50 DEG C) can be determined by mode as described above
In the presence of.
Above-mentioned term is " being higher than the signal detected at a temperature of the more than one detection of particular detection temperature and above-mentioned
The difference between signal detected at a temperature of particular detection " includes the difference that obtains between signal at a temperature of 2 detections.
A signal to detect in the detection temperature for being higher than above-mentioned particular detection temperature in above-mentioned signal, another is upper
State the signal detected at a temperature of particular detection.In the case where being higher than detection temperature 2 or more of above-mentioned particular detection temperature,
It can get more than two differences.
Above-mentioned term " signal detected at a temperature of more than one detection and detects at a temperature of particular detection
Difference between signal " not only includes the difference between the signal detected at a temperature of 2 detections, but also including the use of 2
The difference of difference and the signal acquisition detected at a temperature of other detections between a detection temperature.
With selecting a property, according to an example, using in the more than one inspection for being higher than above-mentioned particular detection temperature (60 DEG C)
The signal detected and the signal detected at a temperature of above-mentioned particular detection carry out Mathematical treatment to obtain under testing temperature (72 DEG C)
The difference obtained, the presence of the target nucleic acid sequence depending on particular detection temperature (60 DEG C).In high above-mentioned detection temperature (72
DEG C) under do not detect signal in the case where, in order to calculate above-mentioned difference, can be used background signal.
According to an example, signal is not detected at the detection temperature (72 DEG C) for being higher than above-mentioned particular detection temperature (60 DEG C)
In the case where, it is contemplated that signal is not detected at the detection temperature (72 DEG C) for being higher than above-mentioned particular detection temperature (60 DEG C), benefit
The signal that detects under above-mentioned particular detection temperature (60 DEG C) determines the target with above-mentioned particular detection temperature (60 DEG C)
The presence of nucleic acid sequence.
It is assumed that if detecting signal under above-mentioned highest detection temperature (72 DEG C), can be identified as having it is above-mentioned relatively most
The target nucleic acid sequence of the Supreme People's Procuratorate's testing temperature exists.
According to an example, the target nucleic acid with above-mentioned particular detection temperature is determined in above-mentioned at least three target nucleic acid sequence
In the presence of sequence, using the signal detected at a temperature of next detection higher than above-mentioned particular detection temperature and
The difference between signal detected at a temperature of above-mentioned particular detection determines the target nucleic acid sequence with above-mentioned particular detection temperature
The presence of column.
According to an example, using the step of above-mentioned difference including the use of to next higher than above-mentioned particular detection temperature
The signal detected at a temperature of detection and the signal detected at a temperature of above-mentioned particular detection carry out Mathematical treatment to obtain
The step of difference.
According to an example, the case where not detecting signal at a temperature of next detection higher than above-mentioned particular detection temperature
Under, it include considering not detect at a temperature of first detection for being higher than above-mentioned particular detection temperature using the step of above-mentioned difference
The step of utilizing the signal detected at a temperature of above-mentioned particular detection the case where signal out.
For example, in the case that determine have above-mentioned 40 DEG C detection temperature target nucleic acid sequences there are, using next
Higher than the signal detected at the detection temperature (50 DEG C) of above-mentioned particular detection temperature (40 DEG C) and in above-mentioned particular detection temperature
Under difference between the signal that detects determine the presence of the target nucleic acid sequence with above-mentioned particular detection temperature.
According to an example, in order to determine that the signal detected at a temperature of above-mentioned 50 DEG C and 40 DEG C detections indicates whether have
The presence of the target nucleic acid sequence of above-mentioned 50 DEG C and 40 DEG C detection temperature, and need a reference value.
An example according to the present invention, according to the method for obtaining above-mentioned difference, in order to analyze the difference of above-mentioned acquisition
It indicates whether the presence with the target nucleic acid sequence of above-mentioned particular detection temperature, threshold value can be used.
An example according to the present invention can be to utilize the above-mentioned difference obtained according to the method for obtaining above-mentioned difference
It is different itself mode determine have above-mentioned particular detection temperature target nucleic acid sequence presence.For example, in order to directly utilize difference
Itself, reflects threshold value during obtaining above-mentioned difference in advance.
An example according to the present invention uses a reference value to determine the presence of target nucleic acid sequence.
In particular instances, it can be indicated by a reference value based on the target nucleus with the detection temperature higher than particular detection temperature
The above-mentioned particular detection temperature that acid sequence provides and the difference higher than the signal at a temperature of the detection of above-mentioned particular detection temperature.
In particular instances, for based on the target nucleic acid sequence offer with the detection temperature higher than particular detection temperature
Above-mentioned particular detection temperature and to the different situation of signal at a temperature of being higher than the detection of above-mentioned particular detection temperature
A reference value compared with for a reference value of the identical situation of above-mentioned 2 signals, difference can for 0.5%, 1%, 1.5%, 2%,
2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%,
10%, 12%, 15%, 20% or 30% or more.
In particular instances, for have be higher than from 2 detection at a temperature of the calculated above-mentioned particular detection temperature of signal
The a reference value of the target nucleic acid sequence of the detection temperature of degree is on duty compared with a reference value of the situation identical for above-mentioned 2 signals
It is different be 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%,
7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 12%, 15%, 20% or 30% or more when, can will for have be higher than spy
The a reference value of the target nucleic acid sequence of the detection temperature of regular inspection testing temperature, which is used in, determines there is above-mentioned specific low temperature detection temperature
The presence of target nucleic acid sequence.
According to an example, when carried out to determine whether using a reference value it is above-mentioned relatively when, division meter signal-based
It counts in stating a reference value.According to an example, in order to determine whether using said reference value and the method for calculating benchmark value can in order to
Detect above-mentioned target nucleic acid sequence and the method for calculating benchmark value is identical or different.
According to an example, the letter for multiple target nucleic acid sequences is detected at a temperature of the detection higher than particular detection temperature
In the case where number, made by considering the pattern for the signal intensity at a temperature of the detection with change of multiple target nucleic acid sequences
With a reference value for being directed to multiple target nucleic acid sequences.
It is in the presence of there is the target nucleic acid sequence higher than the detection temperature of above-mentioned particular detection temperature according to an example
Determining has the presence of the target nucleic acid sequence of above-mentioned particular detection temperature and uses said reference value.
It include higher than upper in the presence of the case where target nucleic acid sequence with the detection temperature for being higher than above-mentioned particular detection temperature
The case where detecting significant signal at a temperature of the detection of particular detection temperature is stated, above-mentioned significant signal is higher than for indicating to have
State the presence of the target nucleic acid sequence of the detection temperature of particular detection temperature.
According to an example, in the absence of there is the target nucleic acid sequence higher than the detection temperature of above-mentioned particular detection temperature,
In order to determine the presence of the target nucleic acid sequence with above-mentioned particular detection temperature, and selectively use said reference value.
It include only detecting in the presence of the case where target nucleic acid sequence with the detection temperature for being higher than above-mentioned particular detection temperature
There is the case where signal of the intensity similar with background signal.
For whole combinations of above-mentioned target nucleic acid sequence or a part, selectively combination can prepare said reference value in advance.
An example according to the present invention, said reference value are the difference between the signal at a temperature of the detection obtained from said combination.
An example according to the present invention can provide at the end of reaction and obtain base under the sufficient reaction condition of saturation signal
Quasi- value.For example, in order to obtain be directed to 2 target nucleic acid sequences combined a reference value, may be selected with above-mentioned each target nucleic acid sequence
The identical condition of the content of column, to provide the saturation signal for being directed to each target nucleic acid sequence.An example according to the present invention, upper
The difference stated between the above-mentioned signal obtained in a reference value calculating process has particular range, in above-mentioned particular range in selection
It states a reference value or selects said reference value referring to above-mentioned particular range.
In view of method for obtaining difference and actually detected as a result, in order to determine the difference that obtains in said sample
Conspicuousness, can be used said reference value.Said reference value can be used in order to obtain above-mentioned difference in the sample, it is above-mentioned as a result,
A reference value participates in determining the existing process of target nucleic acid sequence.
An example according to the present invention, in order to determine the presence of the target nucleic acid sequence with above-mentioned particular detection temperature, In
The above-mentioned base obtained as follows is utilized in the above-mentioned method using at least three target nucleic acid sequence in different detection temperature detection samples
Quasi- value at least one a reference value, (i) in other reaction vessels different from the reaction vessel in above-mentioned steps (a) with phase
Signal-the generation mechanism answered cultivates all of the target nucleic acid sequence with the detection temperature higher than above-mentioned particular detection temperature together
Combination, (ii) are detected at a temperature of being higher than 1 or more the detection temperature and above-mentioned particular detection of above-mentioned particular detection temperature
After signal, (iii) pass through find out at a temperature of being higher than 1 or more detection of above-mentioned particular detection temperature the signal that detects with
And the difference between the signal detected at a temperature of above-mentioned particular detection obtains said reference value.
According to the present invention detect at least three target nucleic acid sequence in the case where, can by with for signal detection and select
2 detection temperature multiple combinations consider the multiple combinations of target nucleic acid sequence together to obtain a variety of a reference values.
An example according to the present invention, from the target nucleic acid sequence with above-mentioned highest detection temperature to above-mentioned minimum inspection
The target nucleic acid sequence of testing temperature is determined the presence of above-mentioned target nucleic acid sequence by their detection temperature sequence, is deposited according to determining target
Method be appropriately selected a reference value.
In above-mentioned example, it is used to determine depositing for the target nucleic acid sequence with above-mentioned particular detection temperature (40 DEG C) to obtain
A reference value, with signal-generation mechanism culture have higher than above-mentioned particular detection temperature (40 DEG C) detection temperature (60 DEG C and
50 DEG C) target nucleic acid sequence whole combinations (that is, target nucleic acid sequence with above-mentioned 60 DEG C detection temperature, with above-mentioned 50 DEG C
Detect the target nucleic acid sequence of temperature and detect the combination of the target nucleic acid sequence of temperature with above-mentioned 60 DEG C and 50 DEG C), not only in height
In (60 DEG C and 50 DEG C) detection signals of 1 or more detection temperature of above-mentioned particular detection temperature (40 DEG C), but also above-mentioned
Signal is detected at particular detection temperature (40 DEG C), then, using in 1 or more detection temperature for being higher than above-mentioned particular detection temperature
The signal detected under degree and the signal detected at a temperature of above-mentioned particular detection obtain the difference between above-mentioned signal.Root
According to identical method, obtaining has depositing for other above-mentioned target nucleic acid sequences for detecting temperature (that is, 60 DEG C and 50 DEG C) for determining
A reference value.
With selecting a property, for the presence of the determining target nucleic acid sequence with above-mentioned particular detection temperature, the above method is also sharp
With at least one a reference value in a reference value obtained as follows, that is, (i) is different from the reaction vessel in above-mentioned steps (a)
Culture has the target of the detection temperature higher than above-mentioned particular detection temperature together with corresponding signal-generation mechanism in reaction vessel
Whole combinations of nucleic acid sequence, (ii) is in next detection temperature and above-mentioned particular detection temperature higher than above-mentioned particular detection temperature
After detecting signal under degree, (iii) passes through to be detected at a temperature of finding out the detection next higher than above-mentioned particular detection temperature
Difference between signal out and the signal detected at a temperature of above-mentioned particular detection obtains a reference value.
In above-mentioned example, it is used to determine depositing for the target nucleic acid sequence with above-mentioned particular detection temperature (40 DEG C) to obtain
A reference value, have with the signal for generating signal-generation mechanism culture higher than above-mentioned particular detection temperature (40 DEG C)
Whole combinations of the target nucleic acid sequence of temperature (60 DEG C and 50 DEG C) are detected (that is, the target nucleic acid sequence with above-mentioned 60 DEG C detection temperature
Column, the target nucleic acid sequence that temperature is detected with above-mentioned 50 DEG C and the target nucleic acid sequence that temperature is detected with above-mentioned 60 DEG C and 50 DEG C
Combination), next higher than at the detection temperature (50 DEG C) of above-mentioned particular detection temperature (40 DEG C) and in above-mentioned particular detection temperature
After detecting signal under degree (40 DEG C), using being detected at next detection temperature (50 DEG C) higher than above-mentioned particular detection temperature
Signal out and the difference between the signal detected under above-mentioned particular detection temperature (40 DEG C) come obtain above-mentioned signal it
Between difference.It is obtained by identical method for determining the above-mentioned target nucleus with other detections temperature (that is, 60 DEG C and 55 DEG C)
The existing a reference value of acid sequence.
An example according to the present invention is being higher than 1 detection temperature of above-mentioned particular detection temperature and above-mentioned by calculating
The ratio or difference between signal detected at a temperature of detection, to obtain said reference value.An example according to the present invention is led to
It crosses and calculates for the signal detected at a temperature of 1 detection higher than above-mentioned particular detection temperature in above-mentioned particular detection temperature
The ratio of the signal detected under degree obtains said reference value.An example according to the present invention, by calculating for above-mentioned
The signal of the signal detected at a temperature of particular detection detected at a temperature of 1 detection higher than above-mentioned particular detection temperature
Ratio obtain said reference value.
According to an example, obtained utilizing for determining a reference value of the target nucleic acid sequence with above-mentioned particular detection temperature
It is available from the special datum value or a part in a reference value that the target nucleic acid sequence of multiple combinations obtains in the case where obtaining threshold value
A reference value obtains above-mentioned threshold value.For example, when analyzing as in the target nucleus with the detection temperature higher than above-mentioned particular detection temperature
When 1 target nucleic acid sequence being not present in acid sequence, use to determine above-mentioned threshold value from not including the upper of 1 target nucleic acid sequence
State a reference value that the combination of target nucleic acid sequence obtains.
In one example, in the case where detecting at least three target nucleic acid sequence, in order to confirm with particular detection temperature
The presence of target nucleic acid sequence is being prepared in advance comprising the target nucleus other than the target nucleic acid sequence with above-mentioned particular detection temperature
After the combination standard sample of acid sequence, a reference value is obtained.In view of said reference value, there is above-mentioned particular detection in order to determine
The presence of the target nucleic acid sequence of temperature and obtain threshold value.
According to an example, using including obtaining for determining the step of the signal detected at a temperature of above-mentioned particular detection
There is the step of existing limit value of the target nucleic acid sequence of above-mentioned opposite highest detection temperature, wrapped using the step of above-mentioned difference
Including acquisition has the step of existing limit value of target nucleic acid sequence of above-mentioned particular detection temperature for determination.
An example according to the present invention has above-mentioned particular detection for determining using the step of above-mentioned difference including acquisition
The step of existing limit value of the target nucleic acid sequence of temperature, when (i) is in the 1 detection temperature for being higher than above-mentioned particular detection temperature
The signal detected in degree and the signal detected at a temperature of above-mentioned particular detection carry out Mathematical treatment or (ii) is being higher than
When not detecting signal at a temperature of 1 or more detection of above-mentioned particular detection temperature, it is contemplated that be higher than above-mentioned particular detection
Signal is not detected at a temperature of 1 or more detection of temperature, is obtained using the signal detected at a temperature of above-mentioned particular detection
Obtain above-mentioned limit value.
An example according to the present invention, it is first determined after the presence with the target nucleic acid sequence of opposite highest detection temperature,
By arrange in descending order successively determine have it is relatively low detection temperature target nucleic acid sequence it is existing in a manner of carry out above-mentioned steps
(b)。
According to an example, above-mentioned 1 reaction vessel further includes at least one attachment device, above-mentioned at least one attachment device point
It does not include additional at least three signal-production for detecting the target nucleic acid sequence other than above-mentioned at least three target nucleic acid sequence
Life structure is mutually distinguishable the above-mentioned signal generated by each device of at least three signal-generation mechanism in said vesse, by
The detector of different types detects above-mentioned signal respectively.
An example according to the present invention, above-mentioned target nucleic acid sequence include nucleotide diversity (in particular, nucleotide polymorphisms).
IV. 2 target nucleic acid sequences are detected using different detection temperature and melting analysis
In another embodiment of the present invention, the present invention, which is provided, detects sample using different detection temperature and melting analysis
The method of interior 2 target nucleic acid sequences, comprising: step (a), in 1 reaction vessel and for detecting above-mentioned 2 target nucleic acid sequences
2 signal-generation mechanisms cultivate said sample together, and using the detector of single type detect caused by signal, borrow
Corresponding signal-generation mechanism is helped to detect each above-mentioned target nucleic acid sequence, one of above-mentioned 2 target nucleic acid sequences has by phase
The relatively high detection temperature that the signal answered-generation mechanism determines, another has by corresponding above-mentioned signal-generation mechanism
Determining relatively low detection temperature, above-mentioned relatively high detection temperature are that can generate for above-mentioned relatively high detection
The temperature of the signal of the target nucleic acid sequence of temperature, above-mentioned relatively low detection temperature be can generate for have it is above-mentioned relatively
The signal of the signal of the target nucleic acid sequence of low detection temperature and the target nucleic acid sequence with above-mentioned relatively high detection temperature
Temperature, the signal generated by above-mentioned 2 signals-generation mechanism are not distinguished by the detector of above-mentioned single type, above-mentioned opposite
Compared with carrying out above-mentioned detection at a temperature of high detection；Step (b), in order to determine the target nucleic acid sequence with above-mentioned relatively low detection temperature
The presence of column, prescribed limit at a temperature of, carry out the melting analysis to the cultivation results object of above-mentioned steps (a)；And step
(c), the target nucleic acid sequence with above-mentioned relatively high detection temperature is determined according to the signal detected in above-mentioned steps (a)
In the presence of utilizing melting analysis result to determine that the target nucleic acid sequence with relatively low detection temperature being deposited in above-mentioned steps (b)
In.
Step (a): with the culture of signal-generation mechanism and signal detection
Firstly, in a reaction vessel with for detect above-mentioned 2 target nucleic acid sequences signal-generation mechanism together with
After cultivating the sample to be analyzed, generated signal is detected using the detector of single type.By above-mentioned 2 signal-generations
The signal that mechanism generates is not distinguished by the detector of above-mentioned single type.
Each target nucleic acid sequence is detected by corresponding signal-generation mechanism.One of above-mentioned 2 target nucleic acid sequences has
By the relatively high detection temperature that corresponding above-mentioned signal-generation mechanism determines, another has by corresponding above-mentioned letter
Number-the determining relatively low detection temperature of generation mechanism.
An example according to the present invention has above-mentioned phase by the real-time detection method detection using different detection temperature
To the target nucleic acid sequence compared with the Supreme People's Procuratorate's testing temperature, there is the target nucleic acid sequence of above-mentioned relatively low detection temperature by melting analysis detection
Column.
Although at a temperature of above-mentioned relatively high detection carry out above-mentioned steps (a) in detection, it is above-mentioned relatively
Without the detection in above-mentioned steps (a) at a temperature of low detection.
During carrying out melting analysis, the signal generating mechanism that selection can produce signal is used to have relatively low detection temperature
The target nucleic acid sequence of degree.
According to an example, since signal-generation mechanism of above-mentioned melting analysis utilizes the hybridization and denaturation of dimer, thus
Signal can be generated according to the reaction condition of above-mentioned steps (a).
According to an example, for generating the letter of signal for the target nucleic acid sequence with above-mentioned relatively low detection temperature
Number-generation mechanism needs be configured to not generate signal at a temperature of above-mentioned relatively high detection.
In the 4th embodiment of the invention, the target nucleic acid with above-mentioned relatively low detection temperature is directed to for generating
The signal generating mechanism of the signal of sequence can generate signal according to reaction condition in above-mentioned steps (a) or not generate signal.I.e.
Make to generate the signal for the target nucleic acid sequence with above-mentioned relatively low detection temperature under special reaction condition, also due to
Signal detection is carried out at a temperature of relatively high detection in above-mentioned steps (a), because of the signal without detecting above-mentioned generation.
The signal for the target nucleic acid sequence with above-mentioned relatively low detection temperature can be generated in above-mentioned steps (b),
And not Bei Jiance, thus do not need to generate above-mentioned signal in the above-mentioned steps (a).
According to an example, signal-generation mechanism for above-mentioned melting analysis does not include probe based on label or is based on
The cutting of dimer generates the mechanism of signal.Above-mentioned melting analysis utilizes the Tm value of hybrid, thus needs detected by exclusion
Above-mentioned hybrid cutting.When being cut, it is possible to unwinding peak will not be generated or greatly reduce detection sensitivity.And
And the signal based on cutting is likely to become false positive signal in real-time detection method.
Above-mentioned steps are carried out under conditions of at least can produce for the above-mentioned relatively high signal for detecting temperature
(a)。
According to an example, above-mentioned steps (a) is carried out during with the amplification of signal of nucleic acid amplification.
According to an example, above-mentioned steps (a) is carried out during the amplification of signal of free nucleic acid amplification.
According to an example, at least one in signal-generation mechanism is to generate signal in a manner of being formed dependent on dimer
Signal-generation mechanism.
According to an example, based on target nucleic acid sequence and with the detection oligonucleotides of above-mentioned target nucleic acid sequence specific hybrid it
Between dimer formed and generate above-mentioned signal.According to an example, by depend on and above-mentioned target nucleic acid sequence specific hybrid
The dimer that is formed of mode of cutting of mediation oligonucleotides generate above-mentioned signal.
According to an example, at least one in signal-generation mechanism is in a manner of the cutting dependent on detection oligonucleotides
Generate signal-generation mechanism of signal.According to an example, after target nucleic acid sequence and detection oligonucleotide hybridization, by above-mentioned
The cutting of oligonucleotides is detected to generate above-mentioned signal.According to an example, based on dependent on special with above-mentioned target nucleic acid sequence
Property hybridization the mode dimer that is formed of cutting of mediation oligonucleotides generate above-mentioned signal.
According to an example, signal-generation mechanism for above-mentioned each target nucleic acid sequence be based on dependent on it is above-mentioned
The mode of the cutting of the mediation oligonucleotides of target nucleic acid sequence specific hybrid forms signal-generation mechanism of dimer.
According to an example, the signal in a manner of the cutting dependent on above-mentioned detection oligonucleotides generates above-mentioned for having
The target nucleic acid sequence of relatively high detection temperature.
In particular, due to following reason, using dependent on for the target nucleic acid sequence with above-mentioned relatively high detection temperature
The signal generation of the mode of the cutting of the detection oligonucleotides of column is highly beneficial, (i) can be in relatively wide detection temperature range
Selection detection temperature；(ii) higher temperature may be selected compared with hybridization is close to method；And (iii) can be by selecting suitable letter
Number-generation mechanism and reaction condition (for example, selection can not generate the signal without cutting and hybridization signal-generation mechanism or
The condition that selection cuts most detection oligonucleotides), signal is not provided during melting analysis.
Signal-generation mechanism of signal is generated in a manner of the formation (for example, molecular beacon) dependent on above-mentioned dimer
It can also be by providing signal based on the cutting of the 5 '-nucleases dependent on reaction condition.As long as being based on cutting to generate
Signal and use above-mentioned signal-generation mechanism, then above-mentioned signal-generation mechanism can be can be considered and the signal based on cutting is provided
Signal-generation mechanism.
According to an example, signal-generation mechanism for the target nucleic acid sequence with above-mentioned relatively high detection temperature is
Signal-generation mechanism of cutting based on detection oligonucleotides, for the target nucleic acid sequence with above-mentioned relatively low detection temperature
Signal-generation mechanism of column is signal-generation mechanism of the formation based on dimer.
According to an example, signal-generation mechanism for the target nucleic acid sequence with above-mentioned relatively high detection temperature is
Signal-generation mechanism of cutting based on detection oligonucleotides, for the target nucleic acid sequence with above-mentioned relatively low detection temperature
Signal-generation mechanism of column is based on the cutting dependent on the mediation oligonucleotides with above-mentioned target nucleic acid sequence specific hybrid
The dimer that is formed of mode generate signal-generation mechanism of signal.
According to an example, above-mentioned 2 signal-generation mechanisms include identical label, from the signal of above-mentioned label not coverlet
The detector of one type is distinguished.
After cultivating (reaction) together with two signal-generation mechanisms for generating signal, single type is utilized
Detector detects the signal of above-mentioned generation.It can produce the signal for the target nucleic acid sequence with relatively high detection temperature
Relatively high detection at a temperature of carry out above-mentioned detection.
Step (b): melting analysis
Then, in order to determine the presence with the above-mentioned relatively low target nucleic acid sequence for detecting temperature, in prescribed limit
At a temperature of carry out for above-mentioned steps (a) cultivation results object melting analysis.
Property for the convenience of explanation in the present specification, it is only above-mentioned to being carried out after completing the detection in above-mentioned steps (a)
The content of step (b) is illustrated.It is to be appreciated that considering cardinal principle of the invention, above-mentioned steps (a) can carried out
In detection before carry out above-mentioned steps (b).Therefore, the process of above-mentioned steps (b) is carried out before the detection of above-mentioned steps (a)
Also belong to the scope of the present invention.
Above-mentioned steps (b) can a variety of melting analysis mistakes well known to person of an ordinary skill in the technical field through the invention
Journey carries out.In the case where not expressing especially, term " melting analysis (stringency) " used in the present specification
With not only include narrow sense melting analysis and further include hybridization analysis meaning come using.The melting analysis of narrow sense refers in basis
The method that temperature adjusts the increased dissociation (dissociation) for detecting dimer under strict conditions.The hybridization analysis of narrow sense
Refer to according to temperature adjust reduce it is stringent under conditions of detection dimer combination (association) method.Not
In the case where especially expressing, term " melting curve (melting curve) " used in the present specification or " unwinding peak song
Line (melting peak curve) " is to include the melting curve obtained from the melting analysis of narrow sense or unwinding peak curve and also wrap
Include the hybrid curve obtained from hybridization analysis or hybridize peak curve meaning come using.Melting curve or hybrid curve can be as existing
Technology, for example, by U.S. Patent No. No. 6174670 and No. 5789167, Drobyshev etc., Gene 188:45
(1997)；Kochinsky and Mirzabekov Human Mutation 19:343(2002)；Livehits etc.,
J.Biomol.Structure Dynam.11:783(1994)；And Howell etc., Nature Biotechnology 17:87
(1999) method illustrated in obtains.For example, melting curve or hybrid curve can be by utilizing hybridization stringency
(stringency) graph making (graphic plot) or display of output signal (output signal) variation of parameter
(display) it constitutes.Output signal can directly draw to Crossbreeding parameters.Typically, for example, melting curve or hybridization are bent
Line has the fluorescence for the degree (that is, degree of hybridization) for indicating dimer structure in Y- axis, and having to draw in X- axis has Crossbreeding parameters.
Unwinding (hybridization) tracing analysis is described referring to the disclosure at U.S. Patent No. 8039215 and unwinding is (miscellaneous
Hand over) peak analysis.
Above-mentioned melting analysis utilizes " Tm " value.Term " Tm " used in the present specification means double-stranded nucleic acid molecule
The half of set (population) be dissociated into the melting temperature of single chain molecule.According to the length and G/ of the nucleotide of hybridization
C content determines Tm value.Wallace rule (R.B.Wallace etc., Nucleic Acids Research, 6:3543- can be passed through
3547 (1979)) and nearest-neighbor method (SantaLucia J.Jr. etc., Biochemistry, 35:3555-
3562(1996))；Sugimoto N. etc., Nucleic Acids Res., 24:4501-4505 (1996)) etc. existing method
Calculate Tm value.
According to an example, carried out detecting signal caused by (melting analysis of narrow sense) in a manner of increasing temperature
Above-mentioned steps (b).With selecting a property, detected in a manner of reducing temperature signal caused by (hybridization analysis of narrow sense) come into
Row above-mentioned steps (b).
According to an example, signal-generation mechanism suitable for above-mentioned melting analysis further include prescribed limit at a temperature of
Any mechanism of signal is generated from the formation of dimer.Also, in signal-generation mechanism for real-time detection method, make
For the mode for substituting melting analysis, the hybridization based on detection oligonucleotides is also used to generate the mechanism of signal.
According to an example, by generated in the way of the formation dependent on dimer signal-generation mechanism of signal come into
Row above-mentioned steps (b).In particular, utilizing with the cutting dependent on the mediation oligonucleotides with above-mentioned target nucleic acid sequence specific hybrid
The dimer that is formed of mode generate signal-generation mechanism of signal to carry out above-mentioned steps (b).
Signal based on the dimer formed in a manner of the cutting dependent on above-mentioned mediation oligonucleotides can be by including
Detection and labeled oligonucleotide cutting and extension-unzipping method (WO 2012/096523), detection and labeled oligonucleotide cutting
And extension-dependent signals are transduceed oligonucleotide hybridization-unzipping method (WO 2013/115442) and detected and label few nucleosides
A variety of methods of acid cutting and the non-Hybridization-dehybridization method (PCT/KR2013/012312) of extension-dependence generate.Above-mentioned detection
With labeled oligonucleotide cutting and extension-unzipping method, detection and labeled oligonucleotide cutting and extension-dependent signals transduction
Oligonucleotide hybridization-unzipping method and detection and labeled oligonucleotide cutting and the non-Hybridization-dehybridization method of extension-dependence are suitable
In be utilized respectively melting analysis based on detection and labeled oligonucleotide cutting and extend method, utilize the detection of melting analysis
With labeled oligonucleotide cutting and extension-dependent signals transduction oligonucleotide hybridization-unzipping method and utilize melting analysis
Detection and labeled oligonucleotide cutting and the non-Hybridization-dehybridization method of extension-dependence.Above-mentioned based on detection and label few nucleosides
In acid cutting and the method extended, signal is not generated using by cutting for the melting analysis in above-mentioned steps (b)
Method.
Also described in pervious patent document above-mentioned detection and labeled oligonucleotide cutting and extension-unzipping method,
Detection and labeled oligonucleotide cutting and extension-dependent signals transduction oligonucleotide hybridization-unzipping method and detection and label
Oligonucleotides cutting and the non-hybridization unzipping method of extension-dependence.
The step of being carried out by above-mentioned detection and labeled oligonucleotide cutting and extension-unzipping method (a)~step (b) is wrapped
Include following step.
Include: step (a), hybridizes target nucleic acid sequence with upstream oligonucleotide and detection and labeled oligonucleotide；Step
(b), under conditions of cutting above-mentioned detection and labeled oligonucleotide, making the result object of above-mentioned steps (a) and there are 5 ' cores
The enzyme of phytase activity is in contact, and above-mentioned upstream oligonucleotide or the induction of its extended chain are based on the enzyme with above-mentioned 5 ' nuclease
Above-mentioned detection and labeled oligonucleotide cutting, this above-mentioned cutting releases comprising above-mentioned detection and labeled oligonucleotide
The segment of a part at 5 '-label positions or 5 '-label positions；Step (c) makes to release from above-mentioned detection and labeled oligonucleotide
Segment with capture and templating oligonucleotides hybridized, from above-mentioned detection and labeled oligonucleotide releasing segment with it is above-mentioned
It captures and the capture position of templating oligonucleotides hybridizes；Step (d), result object and template-dependence using above-mentioned steps (c)
Property nucleic acid polymerase carries out extension, upper by making to hybridize with the capture position of above-mentioned capture and templating oligonucleotides
State segment and extend to form extension dimer, above-mentioned extension dimer have can by the sequence and/or length of (i) above-mentioned segment,
(ii) sequence and/or length of above-mentioned capture and templating oligonucleotides, the sequence of (III) above-mentioned segment and/or length and above-mentioned
It captures and the sequence of templating oligonucleotides and/or the Tm value of length adjustment；Step (e), by a temperature of prescribed limit
To above-mentioned extension dimer carry out unwinding, to provide the existing target signal for indicating above-mentioned extension dimer, according to (i) with it is above-mentioned
At least one that segment and/or above-mentioned capture are connected with templating oligonucleotides marks, (ii) is inserted during above-mentioned extension
Enter the label in above-mentioned extension dimer, (III) is inserted in label in above-mentioned extension dimer during above-mentioned extension
The label being connected with above-mentioned segment and/or above-mentioned capture and templating oligonucleotides or (IV) insertion label
(intercalating label) provides above-mentioned signal；And step (f), extension dimerization is detected by detecting above-mentioned signal
Object, the presence of above-mentioned extension dimer indicate the presence of target nucleic acid sequence.
In this case, above-mentioned detection and labeled oligonucleotide cutting and extension unzipping method further include to follow repeatedly
Mode comprising denaturation between ring is repeated that above-mentioned steps (a)~step (e) is all or part of and step.Above-mentioned
In the step of detection and labeled oligonucleotide cutting and extension-unzipping method (a), substituting above-mentioned upstream oligonucleotide can be used use
In the primer sets of the amplification of target nucleic acid sequence.In this case, above-mentioned detection and labeled oligonucleotide cutting and extension unwinding
Method further include by between iterative cycles comprising denaturation in a manner of be repeated above-mentioned steps (a)~step (f) whole or
The step of a part.
Pass through the above-mentioned step of the invention carried out based on detection and labeled oligonucleotide cutting and extension-unzipping method
(a) step~step (b) includes the following steps, namely.
Include: step (a), hybridizes target nucleic acid sequence with upstream oligonucleotide and detection and labeled oligonucleotide；
Step (b), for making the result object of above-mentioned steps (a) and having under conditions of cutting above-mentioned detection and labeled oligonucleotide
The enzyme of 5 ' nucleases is in contact, and above-mentioned upstream oligonucleotide or the induction of its extended chain are based on above-mentioned 5 ' nuclease
Enzyme above-mentioned detection and labeled oligonucleotide cutting, it includes above-mentioned detection and labeled oligonucleotide that this above-mentioned cutting, which is released,
5 '-label positions or 5 '-label positions a part segment；Step (c) makes to put from above-mentioned detection and labeled oligonucleotide
Segment out with capture and templating oligonucleotides hybridized, from above-mentioned detection and labeled oligonucleotide releasing segment with it is upper
State capture and the capture position hybridization of templating oligonucleotides；Step (d), using above-mentioned steps (c) result object and template-according to
Rely property nucleic acid polymerase to carry out extension, passes through make to hybridize with the capture position of above-mentioned capture and templating oligonucleotides
Above-mentioned segment extends to form extension dimer；Step (e), by detect prescribed limit at a temperature of to depend on above-mentioned extension
Signal that the existing mode of chain generates carries out melting analysis.
Step (c): the presence of target nucleic acid sequence is determined
Finally, being determined using the signal detected in above-mentioned steps (a) with above-mentioned relatively high detection temperature
The presence of target nucleic acid sequence is determined using the melting analysis result in above-mentioned steps (b) with above-mentioned relatively low detection temperature
The presence of the target nucleic acid sequence of degree.
An example according to the present invention, above-mentioned target nucleic acid sequence include nucleotide diversity (in particular, nucleotide polymorphisms).
When combination for real-time detection and based on the signal-generation mechanism for cutting generation signal and is used for melting analysis and base
The letter of signal is generated in the dimer formed in a manner of the cutting dependent on the mediation oligonucleotides hybridized with target nucleic acid sequence
Number-generation mechanism when, can get unexpected result of the invention.In this case, it for melting analysis, should exclude to join
With the signal-generation mechanism of dimer formed and directly generate signal by cutting.
It is noteworthy to be, signal-generation mechanism (example that signal is generated based on cutting to combine as real-time process
Such as, TaqMan method) and as melting analysis is based on detection and labeled oligonucleotide is cut and extension-unzipping method mode
Implement the present invention, to provide very outstanding result.
According to an example, have using the step of the signal detected in above-mentioned steps (a) including acquisition for determination
After the general headquarters of the existing limit value of the target nucleic acid sequence of above-mentioned relatively high detection temperature.
An example according to the present invention, above-mentioned target nucleic acid sequence include nucleotide diversity (in particular, nucleotide polymorphisms).
V. at least three target nucleic acid sequence is detected using different detection temperature and melting analysis
In another embodiment of the present invention, the present invention, which is provided, detects sample using detection temperature analysis and melting analysis
The method of interior at least three target nucleic acid sequence, comprising: step (a), in 1 reaction vessel and for detecting above-mentioned at least three target
At least three signal-generation mechanism of nucleic acid sequence cultivates said sample together, and is produced using the detection of the detector of single type
Raw signal, detects above-mentioned at least three target nucleic acid sequence, above-mentioned at least three target nucleic acid by corresponding signal-generation mechanism respectively
Sequence is respectively provided with the different detection temperature determined by corresponding above-mentioned signal-generation mechanism, and above-mentioned detection temperature is not only
The signal of the target nucleic acid sequence with above-mentioned detection temperature can be generated and can also be generated for higher than above-mentioned detection temperature
The temperature of the signal of the target nucleic acid sequence of the detection temperature of degree, the signal generated by above-mentioned signal-generation mechanism is not by above-mentioned list
The detector of one type is distinguished, and detects a part of target nucleic acid in above-mentioned at least three target nucleic acid sequence by detecting temperature analysis
Sequence, the detection temperature of above-mentioned a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence and be higher than above-mentioned inspection
1 or more detection temperature of testing temperature carries out above-mentioned detection；Step (b), in order in above-mentioned at least three target nucleic acid sequence
The presence for determining other target nucleic acid sequences other than above-mentioned a part of target nucleic acid sequence, prescribed limit at a temperature of, it is right
The cultivation results object of above-mentioned steps (a) carries out melting analysis；And step (c), (i) basis detects in above-mentioned steps (a)
Signal determine the presence of above-mentioned a part of target nucleic acid sequence in above-mentioned target nucleic acid sequence, when in above-mentioned at least three target nucleic acid
In above-mentioned a part of target nucleic acid sequence of sequence determine have particular detection temperature target nucleic acid sequence there are when, then according to exist
Higher than the signal detected at a temperature of 1 or more detection of above-mentioned particular detection temperature and at a temperature of above-mentioned particular detection
Difference between the signal detected determines the presence with the target nucleic acid sequence of above-mentioned particular detection temperature, in above-mentioned detection temperature
When above-mentioned particular detection temperature is opposite highest detection temperature in degree, according to the signal detected at a temperature of above-mentioned particular detection
Determine have above-mentioned particular detection temperature target nucleic acid sequence presence, (ii) in above-mentioned steps (b), according to melting analysis
As a result other target nucleic acid sequences other than above-mentioned a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence are determined
Presence.
The present invention is based on the principles of above-mentioned first embodiment of the invention to the 4th embodiment, therefore, in order to keep away
The excessive repetition for exempting from the complexity for leading to this specification, omits the common content between them.
Step (a): with the culture of signal-generation mechanism and signal detection
Firstly, at least three signal-generation in 1 reaction vessel and for detecting above-mentioned at least three target nucleic acid sequence
After the sample to be analyzed is cultivated together by mechanism, generated signal is detected using the detector of single type.By above-mentioned at least 3
The signal that a signal-generation mechanism generates is not distinguished by the detector of above-mentioned single type.
One in above-mentioned target nucleic acid sequence has opposite highest detection temperature.It can be provided at a temperature of opposite highest detection
Signal-generation mechanism of signal is used to detect the target nucleic acid sequence with opposite highest detection temperature.
In the present invention, a part at least three target nucleic acid sequence is detected by above-mentioned detection temperature analysis, and led to
It crosses melting analysis and detects remainder at least three target nucleic acid sequence.
Term " a part of (some) " includes 1 in sentence " a part of target nucleic acid sequence at least three target nucleic acid sequence "
More than a.In the case where indefinite express, term " detection temperature analysis (detection used in the present specification
Temperature analysis) " refer to the reality including detection at a temperature of different detections for detecting target nucleic acid sequence
When detect.
For generating the signal-generation mechanism for being directed to the signal for the target nucleic acid sequence analyzed by above-mentioned melting analysis
Signal can be generated in above-mentioned steps (a) according to reaction condition or not generate signal.It can generate in above-mentioned steps (b) for logical
The signal for the target nucleic acid sequence that above-mentioned melting analysis is analyzed is crossed, and is detected, therefore do not need in above-mentioned steps (a)
It is middle to generate above-mentioned signal.
Above-mentioned at least three target nucleic acid sequence needs to be utilized respectively signal-generation mechanism with different detection temperature.
According to an example, since signal-generation mechanism for above-mentioned melting analysis utilizes the hybridization and denaturation of dimer,
Thus signal can be generated according to the reaction condition in above-mentioned steps (a).
According to an example, it is being suitble to generate at least 1 for the target nucleic acid sequence detected by above-mentioned detection temperature analysis
Above-mentioned steps (a) is carried out under conditions of a signal.
According to an example, after considering to detect temperature, selection passes through the above-mentioned target nucleic acid to be analyzed of detection temperature analysis
Sequence.In particular, selecting one by detection temperature sequence after selection has the target nucleic acid sequence of above-mentioned highest detection temperature first
Sub-sequence.
According to an example, above-mentioned steps (a) is carried out during with the amplification of signal of nucleic acid amplification.
According to an example, above-mentioned steps (a) is carried out during the amplification of signal of free nucleic acid amplification.
According to an example, at least one in signal-generation mechanism is to generate letter in a manner of the formation dependent on dimer
Number signal-generation mechanism.
According to an example, signal-generation mechanism for above-mentioned each target nucleic acid sequence is produced by the formation of dimer
Signal-generation mechanism of raw signal.
According to an example, based on target nucleic acid sequence and with the detection oligonucleotides of above-mentioned target nucleic acid sequence specific hybrid it
Between the formation of dimer generate above-mentioned signal.According to an example, based on dependent on miscellaneous with above-mentioned target nucleic acid sequence specificity
The dimer that the mode of the cutting of the mediation oligonucleotides of friendship is formed generates above-mentioned signal.
An example according to the present invention, signal-generation mechanism for each above-mentioned target nucleic acid sequence are based on to rely on
Signal-generation machine of dimer is formed in the mode of the cutting of the mediation oligonucleotides with above-mentioned target nucleic acid sequence specific hybrid
Structure.
According to an example, at least one in signal-generation mechanism is the cutting to depend on above-mentioned detection oligonucleotides
Mode generates signal-generation mechanism of signal.According to an example, after above-mentioned detection oligonucleotides hybridizes with target nucleic acid sequence,
By the cutting of above-mentioned detection oligonucleotides, to generate above-mentioned signal.According to an example, to depend on and above-mentioned target nucleic acid sequence
The mode of the cutting of the mediation oligonucleotides of specific hybrid generates above-mentioned signal by detecting the cutting of oligonucleotides.
According to an example, the signal generation in a manner of the cutting dependent on above-mentioned detection oligonucleotides is only used for above-mentioned
The target nucleic acid sequence with opposite highest detection temperature at least three target nucleic acid sequence.
An example according to the present invention, for signal-production of the target nucleic acid sequence with above-mentioned opposite highest detection temperature
Life structure is signal-generation mechanism of the cutting based on detection oligonucleotides, for signal-generation machine of other target nucleic acid sequences
Structure is signal-generation mechanism of the formation based on dimer.
An example according to the present invention, for signal-production of the target nucleic acid sequence with above-mentioned opposite highest detection temperature
Life structure is signal-generation mechanism of the cutting based on detection oligonucleotides, for signal-generation machine of other target nucleic acid sequences
Structure is based on the dimer in a manner of the cutting dependent on the mediation oligonucleotides with above-mentioned target nucleic acid sequence specific hybrid
Formation generate signal.
For example, generating the letter for the target nucleic acid sequence with above-mentioned opposite highest detection temperature by TaqMan method
Number, believed by detection and labeled oligonucleotide cutting and extension method, detection and labeled oligonucleotide cutting and extension-dependence
Number transduction oligonucleotide hybridization method or detection and labeled oligonucleotide cutting and the non-hybridizing method generation of extension-dependence are directed to
The signal of other target nucleic acid sequences.
An example according to the present invention, above-mentioned at least three signal-generation mechanism includes identical label, from above-mentioned label
Signal not by above-mentioned single type detector distinguish.
In a reaction vessel, according to the present invention the quantity of detected above-mentioned target nucleic acid sequence include 3,4,5,6,7,
8,9 and 10 or more target nucleic acid sequences, but not limit and this.
After cultivating (reaction) sample together at least three signal-generation mechanism for generating signal, single type is utilized
Detector detects the signal of above-mentioned generation.According to an example, above-mentioned at least three target nucleic acid sequence is detected by detection temperature analysis
In a part of target nucleic acid sequence, the detection temperature of above-mentioned a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence
Degree and higher than carrying out above-mentioned detection at a temperature of 1 or more detection of above-mentioned detection temperature.
According to an example, signal is detected at a temperature of in order to which whole needed for carrying out above-mentioned detection temperature analysis detects.
The signal of above-mentioned generation is detected using the detector of single type.Above-mentioned at least three target nucleic acid sequence is respectively provided with
The different detection temperature determined by corresponding above-mentioned signal-generation mechanism.Above-mentioned detection temperature is can not only to generate tool
There is the signal of the target nucleic acid sequence of above-mentioned detection temperature, but also can generate for the detection higher than above-mentioned detection temperature
The temperature of the signal of the target nucleic acid sequence of temperature.
According to an example, the target nucleic acid sequence detected by above-mentioned detection temperature analysis, which has, to be higher than through above-mentioned unwinding
The detection temperature of the detection temperature for the target nucleic acid sequence that analysis detection goes out.
Step (b): melting analysis
In above-mentioned at least three target nucleic acid sequence, in order to determine other other than above-mentioned a part of target nucleic acid sequence
The presence of target nucleic acid sequence, prescribed limit at a temperature of carry out for above-mentioned steps (a) cultivation results object melting analysis.
In the present specification, property for the convenience of explanation, it is only above-mentioned to being carried out after completing the detection in above-mentioned steps (a)
The content of step (b) is illustrated.It is to be appreciated that considering cardinal principle of the invention, above-mentioned steps (a) can carried out
In detection before carry out above-mentioned steps (b).Therefore, the process of above-mentioned steps (b) is carried out before the detection of above-mentioned steps (a)
Also belong to the scope of the present invention.
According to an example, by generated in the way of the formation dependent on dimer signal-generation mechanism of signal come into
Row above-mentioned steps (b).In particular, carrying out above-mentioned steps according to based on detection and labeled oligonucleotide cutting and extension-unzipping method
(b)。
Step (c): the presence of target nucleic acid sequence is determined
Finally, melting analysis result using the signal detected in above-mentioned steps (a) and in above-mentioned steps (b) come
Determine the presence of above-mentioned at least three target nucleic acid sequence in sample.
A part of target nucleic acid sequence in above-mentioned target nucleic acid sequence is determined using the signal detected in above-mentioned steps (a)
The presence of column.Determine that there is particular detection temperature in above-mentioned a part of target nucleic acid sequence of above-mentioned at least three target nucleic acid sequence
Target nucleic acid sequence in the presence of, according to be higher than above-mentioned particular detection temperature more than one detection at a temperature of examine
Difference between the signal measured and the signal detected at a temperature of above-mentioned particular detection determines there is above-mentioned particular detection temperature
The presence of the target nucleic acid sequence of degree is the opposite highest detection temperature in above-mentioned detection temperature in above-mentioned particular detection temperature
In the case where, being determined according to the signal detected at a temperature of above-mentioned particular detection has the above-mentioned target of above-mentioned particular detection temperature
The presence of nucleic acid sequence.
According to an example, in order to determine the presence of the target nucleic acid sequence with above-mentioned particular detection temperature, in the above method
It is middle to utilize a reference value obtained as follows, that is, (i) is in other reaction vessels different from the reaction vessel in above-mentioned steps (a)
In together with signal-generation mechanism culture have higher than above-mentioned particular detection temperature detection temperature target nucleic acid sequence own
Combination, (ii) detection letter at a temperature of being higher than 1 or more the detection temperature and above-mentioned particular detection of above-mentioned particular detection temperature
After number, (iii) pass through find out at a temperature of being higher than 1 or more detection of above-mentioned particular detection temperature the signal that detects and
The difference between signal that detects at a temperature of above-mentioned particular detection obtains a reference value.
With selecting a property, for the presence of the determining target nucleic acid sequence with above-mentioned particular detection temperature, in the above-mentioned methods
Also utilize a reference value obtained as follows, that is, (i) is in other reaction vessels different from the reaction vessel in above-mentioned steps (a)
In together with signal-generation mechanism culture have higher than above-mentioned particular detection temperature detection temperature target nucleic acid sequence own
Combination, (ii) detection letter at a temperature of being higher than 1 or more the detection temperature and above-mentioned particular detection of above-mentioned particular detection temperature
After number, (iii) pass through find out the signal detected at a temperature of the detection next higher than above-mentioned particular detection temperature and
The difference between signal that detects at a temperature of above-mentioned particular detection obtains a reference value.
According to an example, in order to obtain be directed to the target nucleic acid sequence detected by above-mentioned detection temperature analysis signal or
Difference between a reference value, at a temperature of needing for the detection of the target nucleic acid sequence detected by above-mentioned melting analysis
When signal detection, can at a temperature of for the detection of the target nucleic acid sequence detected by above-mentioned melting analysis collecting signal.
Passing through the above-mentioned of above-mentioned detection temperature analysis determination according to the determination of the result of the melting analysis in above-mentioned steps (b)
The presence of the target nucleic acid sequence other than a part of target nucleic acid sequence at least three target nucleic acid sequence.
An example according to the present invention, it is first determined after the presence with the target nucleic acid sequence of opposite highest detection temperature,
By arrange in descending order successively determine have it is relatively low detection temperature target nucleic acid sequence it is existing in a manner of carry out above-mentioned steps
(b)。
According to an example, determine the target nucleic acid sequence based on above-mentioned detection temperature analysis there are the step of can be used in really
The presence (for example, for selecting a reference value) of the fixed target nucleic acid sequence based on above-mentioned detection temperature analysis.
In the case where detecting above-mentioned at least three target nucleic acid sequence in real time fashion, the signal generation based on cutting is only used
In a target nucleic acid sequence.In order to which other target nucleic acid sequences can in order to improve the efficiency and preparation (readiness) of analysis
Use the method based on detection and labeled oligonucleotide cutting and extension.
An example according to the present invention, above-mentioned target nucleic acid sequence include nucleotide diversity (in particular, nucleotide polymorphisms).
VI. the kit of multiple detection temperature detection target nucleic acid sequences is utilized
In another embodiment of the present invention, the present invention, which provides, utilizes 2 target nucleus in different detection temperature detection samples
The kit of acid sequence, comprising: (a) 2 signal-generation mechanisms, for detecting above-mentioned 2 target nucleic acid sequences, by corresponding
Signal-generation mechanism detects each above-mentioned target nucleic acid sequence, and a sequence of above-mentioned 2 target nucleic acid sequences has by corresponding
The relatively high detection temperature that signal-generation mechanism determines, another sequence have by corresponding above-mentioned signal-generation mechanism
Determining above-mentioned relatively low detection temperature, above-mentioned relatively high detection temperature are that can generate for above-mentioned relatively high
The temperature of the signal of the target nucleic acid sequence of temperature is detected, above-mentioned relatively low detection temperature is that can generate for above-mentioned phase
The letter of signal to the target nucleic acid sequence of lower detection temperature and the target nucleic acid sequence with above-mentioned relatively high detection temperature
Number temperature, the signal generated by above-mentioned 2 signals-generation mechanism is not distinguished by the detector of above-mentioned single type, above-mentioned
Above-mentioned detection is carried out at a temperature of relatively high detection temperature and above-mentioned relatively low detection；And (b) specification, record by
It is named as the method for the invention of the real-time mode I of the detection of 2 target nucleic acid sequences in the sample using different detection temperature.
In another embodiment of the present invention, the present invention, which provides, utilizes different detection temperature analysis sample nucleic acid sequences
The kit of nucleotide polymorphisms genotype, comprising: (a) signal-generation mechanism, for detecting nucleotide polymorphisms equipotential base
Cause detects each above-mentioned nucleotide polymorphisms allele, above-mentioned nucleotide polymorphisms etc. by corresponding signal-generation mechanism
A gene in the gene of position has the above-mentioned relatively high detection temperature determined by corresponding above-mentioned signal-generation mechanism,
Another gene has the above-mentioned relatively low detection temperature determined by corresponding above-mentioned signal-generation mechanism, above-mentioned opposite
It is the signal that can be generated for the nucleotide polymorphisms gene with above-mentioned relatively high detection temperature compared with the Supreme People's Procuratorate's testing temperature
Temperature, above-mentioned relatively low detection temperature are that can generate for the nucleotide polymorphisms with above-mentioned relatively low detection temperature
The temperature of the signal of the signal of gene and the nucleotide polymorphisms gene with above-mentioned relatively high detection temperature, by above-mentioned 2
The signal that a signal-generation mechanism generates is not distinguished by the detector of above-mentioned single type, in above-mentioned relatively high detection temperature
And above-mentioned detection is carried out at a temperature of above-mentioned relatively low detection；And (b) specification, it records and is named as utilizing different detections
The method of the invention of the real-time mode II of the nucleotide polymorphisms genotyping of temperature.
In another embodiment of the present invention, the present invention, which provides, utilizes at least 3 cores in different detection temperature detection samples
Acid sequence kit, comprising: (a) at least three signal-generation mechanism, for detecting above-mentioned at least three target nucleic acid sequence, by
Corresponding signal-generation mechanism detects above-mentioned at least three target nucleic acid sequence respectively, and above-mentioned at least three target nucleic acid sequence has respectively
There are the different detection temperature determined by corresponding above-mentioned signal-generation mechanism, above-mentioned detection temperature is that can not only generate tool
There is the signal of the target nucleic acid sequence of above-mentioned detection temperature and can also generate for the detection temperature higher than above-mentioned detection temperature
The temperature of the signal of the target nucleic acid sequence of degree, the signal generated by above-mentioned signal-generation mechanism is not by the inspection of above-mentioned single type
Device difference is surveyed, above-mentioned detection is carried out at a temperature of each different detections, and (b) specification, records and be named as using not
With the method for the invention of the real-time mode III of the detection of at least three target nucleic acid sequence in the sample of detection temperature.
In another embodiment of the present invention, the present invention, which is provided, detects sample using different detection temperature and melting analysis
The kit of interior at least two target nucleic acid sequence, comprising: (a) 2 signal-generation mechanisms, for detecting above-mentioned 2 target nucleic acid sequences
Column, detect each above-mentioned target nucleic acid sequence by corresponding signal-generation mechanism, a tool in above-mentioned 2 target nucleic acid sequences
There is the relatively high detection temperature determined by corresponding signal-generation mechanism, another has by corresponding above-mentioned signal-
The above-mentioned relatively low detection temperature that generation mechanism determines, above-mentioned relatively high detection temperature are that can generate for above-mentioned
The temperature of the signal of the target nucleic acid sequence of relatively high detection temperature, above-mentioned relatively low detection temperature are that can generate for tool
There are the signal of the target nucleic acid sequence of above-mentioned relatively low detection temperature and the target nucleic acid with above-mentioned relatively high detection temperature
The temperature of the signal of sequence, the signal generated by above-mentioned 2 signals-generation mechanism is not by the detector area of above-mentioned single type
Not, above-mentioned detection is carried out at a temperature of above-mentioned relatively high detection, for signal-production with above-mentioned relatively high detection temperature
Life structure is signal-generation mechanism of the cutting based on detection oligonucleotides, for above-mentioned relatively low detection temperature
Signal-generation mechanism of target nucleic acid sequence is based on the signal-generation mechanism for forming dimer；And (b) specification, it records
It is named as the of the invention of the real-time mode IV of the detection of 2 target nucleic acid sequences using different detection temperature and melting analysis
Method.
In another embodiment of the present invention, the present invention is provided is detected in sample extremely using detection temperature and melting analysis
The kit of few 3 target nucleic acid sequences, comprising: (a) at least three signal-generation mechanism, for detecting above-mentioned 3 target nucleic acid sequences
Column, detect above-mentioned at least three target nucleic acid sequence, above-mentioned at least three target nucleic acid sequence by corresponding signal-generation mechanism respectively
The different detection temperature determined by corresponding above-mentioned signal-generation mechanism are respectively provided with, above-mentioned detection temperature is can not only
It generates the signal of the target nucleic acid sequence with above-mentioned detection temperature and can also generate for higher than above-mentioned detection temperature
The temperature for detecting the signal of the target nucleic acid sequence of temperature, the signal generated by above-mentioned signal-generation mechanism is not by above-mentioned unitary class
The detector of type is distinguished, and detects a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence by detecting temperature analysis,
The detection temperature of above-mentioned a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence and higher than above-mentioned detection temperature
Above-mentioned detection is carried out at a temperature of one 1 or more detection, for the letter of the target nucleic acid sequence with above-mentioned opposite highest detection temperature
Number-generation mechanism be the cutting based on detection oligonucleotides signal-generation mechanism, for the letter of remaining above-mentioned target nucleic acid sequence
Number-generation mechanism be the formation based on dimer signal-generation mechanism；And (b) specification, it records and is named as utilizing
Detect the method for the invention of the real-time mode V of the detection of at least three target nucleic acid sequence in the sample of temperature and melting analysis.
Kit of the invention is prepared in order to implement the present invention, therefore, in order to avoid leading to the complexity of this specification
Excessive repetition, omit the common content between them.
Above-mentioned all kits of the invention be selectively included buffer, deoxyribonucleic acid polymerase auxiliary because
Sub and deoxyribonucleotide -5- triphosphoric acid etc. expands polymerase chain reaction (for example, polymerase chain reaction for carrying out target
Answer) needed for reagent.Selectively, mentioned reagent box also may include a variety of polynucleotide molecules, reverse transcriptase, a variety of bufferings
Liquid, reagent and for inhibiting the active antibody of deoxyribonucleic acid polymerase.Also, mentioned reagent box may include that progress is positive right
Required reagent when being reacted according to group and negative control group.The optimised quantity of the reagent used in specific reaction can be by institute of the present invention
The those of ordinary skill for belonging to technical field is readily determined.The constituent of mentioned reagent box may be present in independent container or
Multiple constituents may be present in 1 container.
Specification for describing or implementing method of the invention can be stored in suitable storage medium.For example, specification
It can print in the substrate of paper and plastics etc..In another example, specification can be present in read-only memory (CD-ROM) and
The Electronic saving data file mode of the suitable computer readable storage medium of floppy disk etc. exists.In another example, upper
Stating does not include substantive specification in kit, is only to provide the machine for obtaining specification by internet etc. from remote data source
Structure.An example in examples detailed above be comprising browsable specification network address and/or can download specification network address kit.
VII. the storage medium and device of multiple detection temperature detection target nucleic acid sequences are utilized
It is of the invention for implementing in a computer in the storage medium, device and computer program of following description, in order to keep away
The excessive repetition for exempting from the complexity for leading to this specification, omits the common content between them.
In a further embodiment of the invention, the present invention provides computer readable storage medium, comprising at operation
The instruction of device is managed, above-mentioned processor is determined in sample existing for 2 target nucleic acid sequences for executing using different detection temperature
Method, it is above-mentioned to determine that the existing method of 2 target nucleic acid sequences in sample includes: step (a) using different detection temperature, it receives
The signal detected at a temperature of relatively high detection and the signal detected at a temperature of relatively low detection, by corresponding
Signal-generation mechanism detect each above-mentioned target nucleic acid sequence, a sequence of above-mentioned 2 target nucleic acid sequences has by corresponding
The relatively high detection temperature that determines of signal-generation mechanism, another sequence has by corresponding above-mentioned signal-generation machine
Structure determine above-mentioned relatively low detection temperature, above-mentioned relatively high detection temperature be can generate for it is above-mentioned relatively
The temperature of the signal of the target nucleic acid sequence of the Supreme People's Procuratorate's testing temperature, above-mentioned relatively low detection temperature are that can generate for above-mentioned
The signal of the target nucleic acid sequence of relatively low detection temperature and target nucleic acid sequence with above-mentioned relatively high detection temperature
The temperature of signal；And step (b), according to received above-mentioned signal determine the presence of above-mentioned 2 target nucleic acid sequences, (i) root
The target nucleic acid sequence with above-mentioned relatively high detection temperature is determined according to the signal detected at a temperature of above-mentioned relatively high detection
The presence of column, (ii) according to the signal that detects at a temperature of being higher than above-mentioned relatively high detection and it is above-mentioned relatively
Difference between the signal detected at a temperature of low detection determines the target nucleic acid sequence with above-mentioned relatively low detection temperature
In the presence of.
According to an example, in the presence of the target nucleic acid sequence with relatively high detection temperature, in order to determine with above-mentioned
The presence of the target nucleic acid sequence of relatively low detection temperature uses the target nucleic acid sequence with above-mentioned relatively high detection temperature
A reference value.
The a reference value of an example according to the present invention, the target nucleic acid sequence with above-mentioned relatively high detection temperature is stored in
Computer readable storage medium.An example according to the present invention, above-mentioned computer readable storage medium are included in operation this method
The instruction of period input said reference value.An example according to the present invention, above-mentioned computer readable storage medium also include instruction,
Above-mentioned instruction runs the processor for obtaining the method for said reference value.
In another embodiment of the present invention, the present invention provides computer program, runs for determining 2 targets in sample
The processor of the method for nucleic acid sequence and it is stored in computer readable storage medium, 2 target nucleic acid sequences in above-mentioned decision sample
Method include: step (a), receive the signal that detects at a temperature of relatively high detection and in relatively low detection temperature
Under the signal that detects, detect each above-mentioned target nucleic acid sequence by corresponding signal-generation mechanism, utilize the inspection of single type
It surveys device and detects generated above-mentioned signal, one in above-mentioned 2 target nucleic acid sequences has by corresponding signal-generation mechanism
Determining relatively high detection temperature, another has above-mentioned relatively by corresponding above-mentioned signal-generation mechanism determination
Low detection temperature, above-mentioned relatively high detection temperature are that can generate for the target nucleic acid with above-mentioned relatively high detection temperature
The temperature of the signal of sequence, above-mentioned relatively low detection temperature are that can generate for above-mentioned relatively low detection temperature
The temperature of the signal of the signal of target nucleic acid sequence and the target nucleic acid sequence with above-mentioned relatively high detection temperature；And step
(b), according to received above-mentioned signal determine the presence of above-mentioned 2 target nucleic acid sequences, (i) according in above-mentioned relatively high detection
At a temperature of the signal that detects determine the presence with the target nucleic acid sequence of above-mentioned relatively high detection temperature, (ii) is according in height
The signal detected at a temperature of above-mentioned relatively high detection and the signal detected at a temperature of above-mentioned relatively low detection
Between difference determine have it is above-mentioned it is relatively low detection temperature target nucleic acid sequence presence.
In another embodiment of the present invention, the present invention provides computer program, runs for determining 2 targets in sample
The processor of the method for nucleic acid sequence and it is stored in computer readable storage medium.
An example according to the present invention, above-mentioned computer program include the target nucleic acid with above-mentioned relatively high detection temperature
The a reference value of sequence.An example according to the present invention, above-mentioned computer program input above-mentioned base during being included in operation this method
The instruction of quasi- value.An example according to the present invention, above-mentioned computer program also include instruction, and above-mentioned instruction operation is for obtaining
State the processor of the method for a reference value.
In the case where implementing the above method by above-mentioned processor, transport processor by the instruction of starting above procedure
The above-mentioned method of the invention of row.Above procedure instruction may include the instruction for receiving the first signal and second signal, and
May include using received above-mentioned signal determine the existing instructions of above-mentioned 2 target nucleic acid sequences.
It can implement above-mentioned method of the invention in processor, for example, stand-alone computer (stand-alone can be located at
Computer), the computer (network attached computer) or real-time polymerase chain reaction being connected with network
Implement above-mentioned method of the invention in the processor of the transacters such as equipment (data acquisition device).
Above-mentioned computer readable storage medium include imprint CDs (CD-R), read-only memory (CD-ROM), the more function of number
It can CD (DVD), flash memories, floppy disk, hard disk drive, portable hard disk, universal serial bus (USB), tape, mini
Optical disc (MINIDISC), non-volatile memory card, electrically erasable read-only memory (EEPROM), CD (CD), optical storage media, with
The multi storages such as machine access memory (RAM), read-only memory (ROM), system storage and web server.
Relevant to above-mentioned signal data (for example, intensity, amplification cycles number and detection temperature) can by several equipment come
It receives.For example, above-mentioned data can be collected by the processor for being located at polymerase chain reaction transacter.It is being collected
Period can provide in real time to be provided above-mentioned data or can be stored in memory cell or buffer area, can handled after completing experiment
The above-mentioned data in the device Room.Similarly, by the network connection with above-mentioned collection device (for example, local area network (LAN), Virtual Private Network
Network (VPN), internet and Intranet) or be directly connected to (for example, universal serial bus, other direct wired connections or wirelessly connecting
Connect) to the additional system such as stand alone computer system provide above-mentioned data or can Xiang Guangpan, digital versatile disc, floppy disk,
The portable mediums such as portable hard disk or stand alone computer system provide above-mentioned data.Similarly, can by with laptop
Or the network connection of the clients such as desk side computer system is (for example, local area network, Virtual Private Network, internet, Intranet, nothing
Line communication network) to server system provide above-mentioned data set.Above-mentioned signal is detected at a temperature of above-mentioned relatively high detection
In the case where, after receiving or collecting above-mentioned data, provided in above-mentioned data analysis process from for determining target nucleic acid sequence
Existing signal between difference obtain processing signal.Above-mentioned processor is relevant to above-mentioned signal received by handling
Data, to provide the processing signal of difference of the reaction between signal at a temperature of above-mentioned 2 detections.For example, above-mentioned processor
Obtained by the processing received above-mentioned data of institute for the signal that is detected at a temperature of above-mentioned relatively high detection
The ratio of the signal detected at a temperature of above-mentioned relatively low detection.
It may include in flogic system for realizing the instruction for running processor of the invention.Although can to portable hard disk,
Any software storing medium of universal serial bus, floppy disk, CD and digital versatile disc etc. provides above-mentioned instruction, but can
It is downloaded and can be stored in memory module (for example, hard disk drive, local or attachment random access memory, read-only depositing
Other memories such as reservoir).Computer code for running of the invention is operable to such as C, C++, Java, Visual
The Multiple Codes language such as Basic, VBScript, JavaScript, Perl and XML.Also, multilingual and agreement can be used in
Outside and storage inside and transmission based on data of the invention and order.
In another embodiment of the present invention, the present invention, which provides, utilizes target nucleic acid sequence in different detection temperature detection samples
The device of column, the above-mentioned device using target nucleic acid sequence in different detection temperature detection samples includes: (a) computer processor；
And the computer readable storage medium (b) coupled with above-mentioned computer processor.
According to an example, above-mentioned apparatus also includes: reaction vessel can accommodate sample and signal-generation mechanism；Temperature is adjusted
Mechanism, for adjusting the temperature of above-mentioned reaction vessel；And/or the detector of single type, it can detect by above-mentioned signal-generation
The signal that mechanism generates.
According to an example, it is above-mentioned relatively high that above-mentioned computer processor only detects the detector of above-mentioned single type
The signal generated by signal-generation mechanism at a temperature of temperature and relatively low detection is detected, but also can be calculated in above-mentioned phase
To compared with the difference between the signal detected at a temperature of high detection and the signal detected at a temperature of above-mentioned relatively low detection.
Above-mentioned processor can be configured to the structure that 1 processor carries out two kinds of behaviors, that is, can carry out the detection at a temperature of 2 detections
And calculate the order of above-mentioned difference.With selecting a property, processor unit can be configured to 2 processors and carry out two kinds of behaviors respectively
Structure.
First necessary feature of above-mentioned apparatus is that above-mentioned apparatus has processor, and above-mentioned processor is for can detect
The signal generated at a temperature of above-mentioned 2 detections.According to an example, when above-mentioned signal produces together with the amplification of above-mentioned target nucleic acid sequence
When raw, above-mentioned apparatus includes processor, and above-mentioned processor in each amplification cycles for that can detect in above-mentioned 2 detections temperature
The lower signal generated of degree.
Second necessary feature of above-mentioned apparatus is that above-mentioned apparatus has processor, and above-mentioned processor passes through to above-mentioned
The signal detected at a temperature of 2 detections is handled to obtain the difference between above-mentioned signal.According to an example, above-mentioned signal it
Between difference digital value is expressed as by Mathematical treatment.
According to an example, above-mentioned processor can in the existing apparatus for detecting target nucleic acid sequence (for example, poly- in real time
Polymerase chain reaction unit) setting software mode realize.According to an example, above-mentioned apparatus can be at a temperature of at least two be detected
Detect signal, and the processor including carrying out Mathematical treatment at least two testing result.
In another embodiment of the present invention, computer readable storage medium is provided, comprising for running processor
Instruction, above-mentioned processor is used to execute the presence that at least three target nucleic acid sequence in sample is determined using different detection temperature, above-mentioned
Include: step (a) to the existing method for determining at least three target nucleic acid sequence in sample using different detection temperature, receives
The signal detected at a temperature of at least three detection, detects above-mentioned at least three target nucleus by corresponding signal-generation mechanism respectively
Acid sequence, above-mentioned at least three target nucleic acid sequence are respectively provided with the different detections determined by corresponding above-mentioned signal-generation mechanism
Temperature, above-mentioned detection temperature are that can not only generate the signal of the target nucleic acid sequence with above-mentioned detection temperature and can also generate
For the temperature of the signal of the target nucleic acid sequence with the detection temperature higher than above-mentioned detection temperature, by above-mentioned signal-generation machine
The signal that structure generates is not distinguished by the detector of above-mentioned single type, carries out above-mentioned detection at a temperature of each different detections；With
And step (b), the presence of above-mentioned at least three target nucleic acid sequence is determined according to received signal, in above-mentioned at least three target nucleic acid
Determined in sequence the target nucleic acid sequence with particular detection temperature there are when, according to be higher than 1 of particular detection temperature with
On detection at a temperature of difference between the signal detected and the signal detected at a temperature of above-mentioned particular detection determine
The presence of target nucleic acid sequence with above-mentioned relatively low detection temperature, the above-mentioned particular detection temperature in above-mentioned detection temperature
When degree is opposite highest detection temperature, above-mentioned target nucleic acid sequence is determined according to the signal detected at a temperature of above-mentioned particular detection
Presence.
An example according to the present invention, a reference value are stored in computer readable storage medium.An example according to the present invention,
Above-mentioned computer readable storage medium includes the instruction that said reference value is inputted during running the above method.It is according to the present invention
One example, above-mentioned computer readable storage medium also include the instruction for realizing processor, and above-mentioned processor operation is for obtaining
The method for stating a reference value.
In another embodiment of the present invention, the present invention provides computer program, and above-mentioned computer program operation carries out
For using it is different detection temperature detection samples at least three target nucleic acid sequence the above method processor and be stored in calculating
Machine readable storage medium storing program for executing or for being stored in computer readable storage medium.
In a further embodiment of the invention, the present invention provides computer readable storage medium, comprising at operation
The instruction of device is managed, above-mentioned processor is used to execute the nucleotide polymorphisms base using different detection temperature to sample nucleic acid sequence
Because of the method that type is analyzed, it is above-mentioned using different detection temperature to the nucleotide polymorphisms genotype of sample nucleic acid sequence
The method analyzed includes: step (a), receives the signal detected at a temperature of relatively high detection and relatively low
The signal detected at a temperature of detection detects each above-mentioned nucleotide polymorphisms equipotential base by corresponding signal-generation mechanism
Cause, a gene in above-mentioned nucleotide polymorphisms allele, which has, to be determined by corresponding above-mentioned signal-generation mechanism
Above-mentioned relatively high detection temperature, another gene are above-mentioned opposite with determining by corresponding above-mentioned signal-generation mechanism
Lower detection temperature, above-mentioned relatively high detection temperature are that can generate for the nucleosides with above-mentioned relatively high detection temperature
The temperature of the signal of sour Genetic polymorphism, above-mentioned relatively low detection temperature are that can generate for above-mentioned relatively low inspection
The signal of the nucleotide polymorphisms gene of testing temperature and with it is above-mentioned it is relatively high detection temperature nucleotide polymorphisms gene
Signal temperature；And step (b), nucleotide polymorphisms genotype is determined according to the difference between received signal.
An example according to the present invention, for by the nucleotide polymorphisms equipotential base with above-mentioned relatively high detection temperature
The homozygote formed by the homozygote of formation and/or the nucleotide polymorphisms allele by detecting temperature with above-mentioned low temperature
And/or a reference value of heterozygote is stored in above-mentioned computer readable storage medium.An example according to the present invention, above-mentioned computer
Readable storage medium storing program for executing includes the instruction that said reference value is inputted during running the above method.An example according to the present invention, on
Stating computer readable storage medium also includes the instruction for realizing processor, and above-mentioned processor operation is for obtaining said reference value
Method.
In another embodiment of the present invention, the present invention provides computer program, and above-mentioned computer program operation is used for
Using it is different detection temperature analysis sample nucleic acid sequences nucleotide polymorphisms genotype the above method processor and deposit
It is stored in computer readable storage medium or for being stored in computer readable storage medium.
An example according to the present invention, above-mentioned computer program include to by the core with above-mentioned relatively high detection temperature
The homozygote and/or the nucleotide polymorphisms equipotential base by detecting temperature with above-mentioned low temperature that nucleotide polymorphism allele is formed
Because of the homozygote of formation and/or a reference value of heterozygote.An example according to the present invention, above-mentioned computer program are included in operation
The instruction of said reference value is inputted during the above method.An example according to the present invention, above-mentioned computer program also include to be used for
The instruction of processor is run, above-mentioned processor carries out the method for obtaining said reference value.
In a further embodiment of the invention, the present invention provides computer readable storage medium, comprising at operation
The instruction of device is managed, above-mentioned processor is determined in sample existing for 2 target nucleic acid sequences for executing using different detection temperature
Method, it is above-mentioned to determine that the existing method of 2 target nucleic acid sequences in sample includes: step (a) using different detection temperature, connect
It receives at a temperature of the determining existing above-mentioned relatively high detection with the above-mentioned relatively high target nucleic acid sequence for detecting temperature
Signal and for determining existing for the target nucleic acid sequence with above-mentioned relatively low detection temperature from the temperature of prescribed limit
Under melting analysis signal, detect above-mentioned each target nucleic acid sequence, above-mentioned 2 target nucleus by corresponding signal-generation mechanism
One in acid sequence has the relatively high detection temperature determined by above-mentioned corresponding signal-generation mechanism, another tool
There is the relatively low detection temperature determined by above-mentioned corresponding signal-generation mechanism, above-mentioned relatively high detection temperature is energy
Enough temperature for generating the signal for the target nucleic acid sequence with above-mentioned relatively high detection temperature, above-mentioned relatively low detection temperature
Degree can be generated for the signal of the target nucleic acid sequence with above-mentioned relatively low detection temperature and for above-mentioned opposite
Compared with the temperature of the signal of the target nucleic acid sequence of the Supreme People's Procuratorate's testing temperature, in order to determine the target nucleic acid with above-mentioned relatively low detection temperature
The presence of sequence, above-mentioned prescribed limit at a temperature of carry out melting analysis；And step (b), according to the received above-mentioned letter of institute
The presence for number determining 2 target nucleic acid sequences, determined according to signal at a temperature of above-mentioned relatively high detection have it is above-mentioned opposite
Compared with the presence of the target nucleic acid sequence of the Supreme People's Procuratorate's testing temperature, determine that there is above-mentioned relatively low inspection according to from the signal of above-mentioned melting analysis
The presence of the target nucleic acid sequence of testing temperature.
In another embodiment of the present invention, the present invention provides computer program, and above-mentioned computer program operation is used for
Processor and storage using the above-mentioned method of 2 target nucleic acid sequences in different detection temperature and melting analysis detection sample
In computer readable storage medium or for being stored in computer-readable storage.
In a further embodiment of the invention, the present invention provides computer readable storage medium, comprising at operation
The instruction of device is managed, above-mentioned processor utilizes at least three target nucleic acid sequence in detection temperature and melting analysis detection sample for executing
The method of column, the above-mentioned method using at least three target nucleic acid sequence in detection temperature and melting analysis detection sample includes: step
(a), above-mentioned at least 3 (i) are received in order to determine the presence of a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence
The detection temperature of a part of target nucleic acid sequence in a target nucleic acid sequence and 1 or more detection temperature higher than above-mentioned detection temperature
Degree under signal, and (ii) in order to determine in above-mentioned at least three target nucleic acid sequence in addition to above-mentioned a part of target nucleic acid sequence
Except other target nucleic acid sequences presence, receive from prescribed limit at a temperature of melting analysis signal, by corresponding
Signal-generation mechanism detect above-mentioned at least three target nucleic acid sequence respectively, above-mentioned at least three target nucleic acid sequence, which is respectively provided with, to be borrowed
The different detection temperature that corresponding above-mentioned signal-generation mechanism determines are helped, above-mentioned detection temperature can not only be generated with upper
It states the signal of the target nucleic acid sequence of detection temperature and can also generate for the detection temperature higher than above-mentioned detection temperature
The temperature of the signal of target nucleic acid sequence detects above-mentioned one in above-mentioned at least three target nucleic acid sequence by analysis detection temperature
Part target nucleic acid sequence, the detection temperature and height of above-mentioned a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence
Above-mentioned detection is carried out at a temperature of 1 or more detection of above-mentioned detection temperature, in order to determine above-mentioned at least three target nucleic acid sequence
In other target nucleic acid sequences other than above-mentioned a part of target nucleic acid sequence presence and carry out the temperature of above-mentioned prescribed limit
Melting analysis under degree；And step (b), it (i) is determined by received above-mentioned signal upper in above-mentioned target nucleic acid sequence
State the presence of a part of target nucleic acid sequence, wherein in above-mentioned a part of target nucleic acid sequence of above-mentioned at least three target nucleic acid sequence
In the presence of determining the target nucleic acid sequence with particular detection temperature, according to higher than the 1 of above-mentioned particular detection temperature
Difference at a temperature of a above detection between the signal detected and the signal detected at a temperature of above-mentioned particular detection is true
Surely the presence of the target nucleic acid sequence with above-mentioned particular detection temperature, when above-mentioned particular detection temperature is in above-mentioned detection temperature
When opposite highest detection temperature, determine that there is above-mentioned particular detection temperature by the signal detected at a temperature of above-mentioned particular detection
The presence of the target nucleic acid sequence of degree, (ii) are determined in above-mentioned at least three target nucleic acid sequence by from the information of above-mentioned melting analysis
Other target nucleic acid sequences other than above-mentioned a part of target nucleic acid sequence presence.
In another embodiment of the present invention, the present invention provides computer program, and above-mentioned computer program operation utilizes
Temperature analysis and melting analysis are detected to detect the processor of the method for at least three target nucleic acid sequence in sample and be stored in calculating
Machine readable storage medium storing program for executing or for being stored in computer readable storage medium.
Feature and advantage of the invention are summarized as follows:
(a) in the present invention using different detection temperature, even if only using the mark of single type in a reaction vessel
Note also can detect multiple target nucleic acid sequences with existing embodiment.In the prior art, after completing target amplification, pass through
Melting analysis detects multiple target nucleic acid sequences.Unlike this, melting analysis is not necessarily to after target amplification due to the present invention, thus greatly
It is big to shorten analysis time.
(b) interestingly the present inventor confirms, and the signal for being directed to target nucleic acid sequence is being generated using signal-generation mechanism
In the case where, (i) according to the type of signal generating mechanism, the detection of signal is adjusted at a certain temperature, (ii) is selected
2 detections at a temperature of when detecting signal, the signal that detects changes according to specific specific pattern at a temperature of above-mentioned 2 detections.
The present inventor is by it is suitable for the detections of target nucleic acid sequence, to realize the present invention.
(c) in the present invention using different detection temperature, by using by dependent on special with target nucleic acid sequence
Property hybridization the mode dimer that is formed of cutting of mediation oligonucleotides provide signal-generation mechanism of signal (for example, base
In the method for detection and labeled oligonucleotide cutting and extension) it can induce unexpected effect.First, using based on detection and mark
Remember that oligonucleotides cutting and the method extended etc. mediate in the method for oligonucleotides, the Tm of the dimer of easily adjustable formation
Value, to be readily able to select detection temperature.Second, when with utilize polymerizeing with 5 '-nucleases for target amplification
Enzyme is utilized together when directly providing the method for signal from the probe hybridized with target nucleic acid sequence, and above-mentioned probe is possible to above-mentioned
The cutting of 5 '-nucleases, thus can have an impact the explanation of signal.As above-mentioned based on detection and labeled oligonucleotide cutting
And the method extended, the mediation oligonucleotides based on 5 '-nucleases is usually utilized using the method for mediating oligonucleotides
Cutting, therefore, the relevant these problems of the final explanation that does not generate usually to signal.Finally, as based on detection and the few core of label
Method thuja acid cutting and extended, in using the method for mediating oligonucleotides, since above-mentioned dimer has and target nucleic acid sequence
Unrelated sequence is arranged, thus the dimer with specific Tm value can be formed.Unlike this, direct with target nucleic acid sequence in utilization
In the method for the probe of hybridization, since at least 1 chain being formed by dimer includes the sequence complementary with target nucleic acid sequence,
Therefore in the case where there is variation on target nucleic acid sequence, the dimer with the Tm value not being intended to can be formed.
(d) in the example of the invention for detecting multiple target nucleic acid sequences using different detection temperature in real time fashion
In, combination (i) for the letter for generating signal in a manner of the cutting dependent on detection oligonucleotides of 1 target nucleic acid sequence
Number-generation mechanism (for example, TaqMan method) and (ii) for other target nucleic acid sequences by depend on and target nucleic acid sequence
The dimer that the mode of the cutting of the mediation oligonucleotides of column specific hybrid is formed provides signal-generation mechanism (example of signal
Such as, the method based on detection and labeled oligonucleotide cutting and extension) it can induce unexpected effect.
For the cutting of above-mentioned detection oligonucleotides, tool is usually utilized using the method for the cutting of above-mentioned detection oligonucleotides
There is the enzyme (in particular, Taq polymerase) of 5 '-nucleases.The existing side of signal is generated in the direct cross by detection probe
In method (for example, method of molecular beacons, hybridization probe method or Hybeacon method), above-mentioned detection probe is had 5 '-nucleic acid
A possibility that enzyme (in particular, Taq polymerase) cutting of enzymatic activity, is very high.The cutting of above-mentioned detection probe is likely to result in because of inspection
Susceptibility caused by the consumption of probing needle reduces (for example, hybridization probe method), and turns using cutting-dependent signals
It can lead to false positive signal (for example, method of molecular beacons) in the method led.Labeled primed method is (for example, sunrise method
Or scorpion shape method) there is no such as above-mentioned detecting probe methods aiming at the problem that cutting, but exist and needed to adjust detection temperature
The shortcomings that adjusting the Tm value of amplified production itself.Reduced value, due to above-mentioned based on detection and labeled oligonucleotide cutting and extension
Method utilize with target nucleic acid sequence specific hybrid mediation oligonucleotides cutting, therefore not by based on have 5 '-nucleic acid
The influence of the enzyme (in particular, Taq polymerase) of enzymatic activity.Also, it is above-mentioned based on detection and labeled oligonucleotide cutting and extend
Method can easily adjust the Tm value for the dimer to be formed, to be readily able to select detection temperature.
(e) real-time detection is utilized to a part of target, melting analysis is utilized to other targets, to examine in 1 reaction vessel
It surveys in an example of the invention for multiple target nucleic acid sequences, the letter for being suitable for the characteristic of target nucleic acid sequence to be analyzed may be selected
Number-generation mechanism, thus, it is possible to multiple target nucleic acid sequences are detected in a manner of more efficiency.
(f) in the example of the invention for detecting multiple target nucleic acid sequences using real-time detection and melting analysis, make
For signal-generation mechanism for the target nucleic acid sequence detected in real time fashion, with the cutting dependent on detection oligonucleotides
Mode generate signal-generation mechanism (for example, TaqMan method) of signal being capable of convenience and efficiency to further increase
Detect multiple target nucleic acid sequences.Detection probe can occur in the method using the cutting of detection oligonucleotides such as TaqMan method
Cutting, in this case, after completing real time reaction, there is no can produce the dimer of signal in melting analysis, by
This, can easily choose the Tm value for other target nucleic acid sequences detected by melting analysis.
(g) in the example of the invention for detecting multiple target nucleic acid sequences by real-time detection and melting analysis, (i) needle
To the signal-generation mechanism (example for generating signal in a manner of the cutting dependent on detection oligonucleotides of a target nucleic acid sequence
Such as, TaqMan method) and (ii) for other target nucleic acid sequences by dependent on miscellaneous with above-mentioned target nucleic acid sequence specificity
The dimer that the mode of the cutting of the mediation oligonucleotides of friendship is formed provides signal-generation mechanism of signal (for example, based on detection
With labeled oligonucleotide cut and extend method) combination can induce unexpected effect.
According to the prior art using the hybridization between target nucleic acid sequence and detection oligonucleotides, exist due to probe cutting
Caused susceptibility is reduced and (is reduced including the susceptibility in melting analysis) and false positive generation etc. because of caused by cutting is tight
The problem of weight.The present invention completely disengages these problems, in the above-mentioned method based on detection and labeled oligonucleotide cutting and extension
In, thus the Tm value of the easily adjustable dimer used to detect can easily choose for real-time detection and use
Detection temperature and the peak Tm value used for melting analysis.
The present invention is described in more details by embodiment.These embodiments are only used for having more the present invention
The explanation of body, disclosed in the claimed range of appended invention the scope of the invention is not limited to these embodiments,
This is obvious for those of ordinary skill in the art to which the present invention belongs.
Embodiment
Embodiment 1: based on the detection and labeled oligonucleotide cutting including the signal detection under different temperatures and extend real
When polymerase chain reaction two targets detection
The present inventor is cut using the detection of 1 sense channel and the signal detection under different temperatures and labeled oligonucleotide
And extends real-time polymerase chain reaction and carried out the experiment that could detect two target nucleic acids in 1 reaction vessel.
For the cutting and detection of the extension of upstream primer and downstream primer, detection and labeled oligonucleotide and the few core of label
The extension of acid fragments, and used the Taq deoxyribonucleic acid polymerase with 5 ' nucleases.By gonococcus (NG,
Neisseria gonorrhoeae) genomic deoxyribonucleic acid and Chlamydia (CT, Chlamydia
Trachomatis genomic deoxyribonucleic acid) is used as target nucleic acid sequence.
In order to detect Chlamydia and gonococcus, and detection and labeled oligonucleotide is used to cut and extend poly- in real time
Polymerase chain reaction.In the presence of target, detection and labeled oligonucleotide are cut, and generate detection and label few nucleosides
Acid fragment.Above-mentioned detection and labeled oligonucleotide segment are annealed at the capture position of capture and templating oligonucleotides, are being caught
It catches and extends on the templating position with templating oligonucleotides and form capture and templating oligonucleotides and extend dimer (two
Polymers captures and templating oligonucleotides).Signal is provided by the formation of above-mentioned extension dimer, is extending dimer-formation
At a temperature of measure signal, thus can get amplification curve.
In the present embodiment, it is contemplated that signal generating mechanism selects " 72 DEG C " to examine as the signal for Chlamydia
Testing temperature selects " 60 DEG C " as the signal detection temperature for being used for gonococcus.According to depositing for above-mentioned Chlamydia or gonococcus
There is the adjustable Tm value adjusted based on its sequence and length in extension dimer generated.In the present embodiment,
The sequence and length of above-mentioned extension dimer be designed to 72 DEG C at a temperature of form above-mentioned dimer signal be provided.It is another
Aspect, sequence and length for the above-mentioned extension dimer of gonococcus be designed to 60 DEG C at a temperature of form above-mentioned dimerization
Object provides signal, but 72 DEG C at a temperature of do not form above-mentioned dimer and be dissociated, to provide different signals.In
At a temperature of 72 DEG C of detection, generates the signal for above-mentioned Chlamydia and detect signal.At a temperature of 60 DEG C of detection,
The signal for being directed to above-mentioned Chlamydia is not only generated, but also generates the signal quilt for above-mentioned gonococcus and detects.
Above-mentioned detection and labeled oligonucleotide and capture and templating oligonucleotides in a manner of preventing its extension by carbon
Spacer region blocks.Above-mentioned capture and templating oligonucleotides are in its 5 '-end by fluorescent reporter (CAL Fluor Red
610) it marks and (sequence 4 and sequence 8) is marked by quenching molecules (BHQ-2) at its templating position.
Prepare to separately include Chlamydia, gonococcus, Chlamydia and the mixture of gonococcus and pair without target
According to 4 reaction tubes of group.
The upstream primer, downstream primer, detection and the labeled oligonucleotide that use in the present embodiment and capture and templating
The sequence of oligonucleotides is as follows.
NG_F 5 '-TACGCCTGCTACTTTCACGCTIIIIIGTAATCAGATG-3 ' (sequence 1)
NG_R 5 '-CAATGGATCGGTATCACTCGCIIIIICGAGCAAGAAC-3 ' (sequence 2)
NG_PTO 5’-GTACGCGATACGGGCCCCTCATTGGCGTGTTTCG [C3 spacer] -3 ' (sequence 3)
NG_CTO 5’-[BHQ-2]TTTTTTTTTTTTTTTTTTTG[T(Cal Fluor Red 610)]
ACTGCCCGTATCGCGTAC [C3 spacer] -3 ' (sequence 4)
Chlamydia _ F 5 '-GAGTTTTAAAATGGGAAATTCTGGTIIIIITTTGTATAAC-3 ' (sequence 5)
CT_R 5 '-CCAATTGTAATAGAAGCATTGGTTGIIIIITTATTGGAGA-3 ' (sequence 6)
CT_PTO 5’-GATTACGCGACCGCATCAGAAGCTGTCATTTTGGCTGCG [C3spacer] -3 ' (sequence 7)
CT_CTO 5’-[BHQ-2]GCGCTGGATACCCTGGACGA[T(Cal Fluor Red 610)]
ATGTGCGGTCGCGTAATC [C3 spacer] -3 ' (sequence 8)
(I: deoxyinosine)
(the 5 '-label positions that underlined text indicates detection and labeled oligonucleotide)
Using including the target nucleic acid (base of the chlamydia trachomatis of the genomic deoxyribonucleic acid and 10pg of the gonococcus of 10pg
Because of the gene of the chlamydia trachomatis of the genomic deoxyribonucleic acid and 10pg of group DNA or the gonococcus of 10pg
The mixture of group DNA), the upstream primer (sequence 1) and 5pmole of the target amplification for gonococcus of 5pmole
Downstream primer (sequence 2), the capture of 3pmole and templating oligonucleotides (sequence 3), 1pmole capture oligonucleotides with
Capture and templating oligonucleotides (sequence 4), 5pmole for chlamydia trachomatis target amplification upstream primer (sequence 5) and
The downstream primer (sequence 6) of 5pmole, the detection of 3pmole and labeled oligonucleotide (sequence 7), 1pmole capture and template
Change oligonucleotides (sequence 8) and 10 μ l 2 × main mixed liquor (it is final, 200 μM of triphosphate deoxy-nucleotide (dNTPs),
The MgCl of 2mM2And the Taq deoxyribonucleic acid polymerase of 2U) the final volume of 20 μ l to have carried out Real-Time Polymerase Chain anti-
It answers.The pipe containing above-mentioned reaction mixture is set to be located at real time thermocycler (CFX96, Bole company (Bio-Rad)), and at 50 DEG C
At a temperature of react after five minutes, 95 DEG C at a temperature of be denaturalized 15 minutes, and with 95 DEG C at a temperature of denaturation 30 seconds,
60 DEG C at a temperature of denaturation 60 seconds, implement repeatedly 50 times in 72 DEG C of at a temperature of 30 seconds modes of denaturation.It is followed each
The detection of signal has been carried out at a temperature of 60 DEG C of ring and 72 DEG C.
As shown in Figure 1A, in the presence of chlamydia trachomatis (pipe (1) and pipe (3)), 72 DEG C at a temperature of detect
Signal is gone out.There is only gonococcus (pipe (2)), 60 DEG C at a temperature of detected signal, but at 72 DEG C
At a temperature of do not detect signal.In the case where target nucleic acid is not present, (pipe (4)) does not detect signal.The result of Figure 1A is shown
Out, the trachoma with above-mentioned relatively high detection temperature is directed in 72 DEG C of the at a temperature of generation as relatively high detection temperature
The signal of Chlamydia, but the signal to gonococcus is not generated.Therefore, it is known that as follows: utilizing above-mentioned relatively high detection temperature
Under signal detection can determine with it is above-mentioned it is relatively high detection temperature chlamydia trachomatis presence.And in pipe (2), lead to
Cross using because signal at a temperature of above-mentioned relatively high detection be not present and relatively low detection at a temperature of above-mentioned signal
The difference in the presence of caused by, it may be determined that the presence of the gonococcus with above-mentioned relatively low detection temperature.
In order to test from have it is above-mentioned it is relatively high detection temperature chlamydia trachomatis signal there are the case where or not
In the presence of whether also can detect the presence of the gonococcus with above-mentioned relatively low detection temperature, and utilize a variety of close
Method calculates the difference between above-mentioned relatively high detection temperature and the signal detected at a temperature of above-mentioned relatively low detection
Different (Figure 1B~Fig. 1 E).
Above-mentioned Figure 1B show the RFU value of the endpoint at a temperature of 72 DEG C and 60 DEG C ratio (all RFU values derive from device
" baseline deduction curve matching (baseline subtracted curve fit) " in software analyzes data, and is sent).
Be 1.2 there is only the ratio of (pipe (1)) when chlamydia trachomatis, but Chlamydia and gonococcus it is equal in the presence of (pipe (3))
Ratio is 2.1.Also, the ratio of (pipe (4)) is respectively when there is only the ratio of (pipe (2)) when gonococcus and without any target nucleic acid
36.7 and 0.7.In order to determine the existence or non-existence of gonococcus, and applicable threshold 1.5.According to above-mentioned threshold value, in pipe (2) and pipe
(3) presence of confirmation gonococcus in, and confirm in pipe (1) and pipe (4) without gonococcus.Consider from only comprising Chlamydia
Final-ratio (End-Raio) determine above-mentioned threshold value.Such as pipe (2), it is being determined as that there is the relatively high sand for detecting temperature
In the case that chlamydia oculogenitale is not present, instead of threshold value 1.5, can independently it set for the above-mentioned letter at a temperature of relatively low detection
Number.
Using 72 DEG C and 60 DEG C at a temperature of signal other close in method, in each circulation calculate 72 DEG C and 60
The ratio of fluorescence signal at a temperature of DEG C, and to above-mentioned ratio (all RFU values source handled to draw of circulation drawing
In device software without baseline deduction (no baseline subtraction) " analysis data, and be sent).For determination
The existence or non-existence of gonococcus has used threshold value 0.1.To consider the side from the ratio drawing for the pipe for only including Chlamydia
Formula determines above-mentioned threshold value.As shown in Figure 1 C, the presence of gonococcus, C are confirmed in pipe (2) and pipe (3)tValue be respectively 32.90 and
33.18.C is not obtained in pipe (1) and pipe (4)tValue.In each cycle, it is deducted from the fluorescence intensity at a temperature of 72 DEG C
Fluorescence intensity at a temperature of 60 DEG C, can be to upper for circulation thus instead of the mode of calculating ratio, and in order to detect target
Result is stated to draw.
It detects using the existing signal of Chlamydia is indicated at a temperature of relatively high detection with above-mentioned phase
To in the another method of the gonococcus of lower detection temperature, in order to analyze the at a temperature of letter that obtains at 60 DEG C in each pipe
Number, and it is applicable in individual threshold.In the case where detecting signal at a temperature of 72 DEG C, pass through each End- at a temperature of 72 DEG C
RFU comes to calculate the individual threshold for the signal at a temperature of 60 DEG C from above-mentioned pipe multiplied by threshold value (1.5).Consider from only comprising sand
Final-ratio (B referring to Fig.1) of the pipe of chlamydia oculogenitale determines above-mentioned threshold value (1.5).72 DEG C at a temperature of do not detect letter
In the case where number, according to the common setting method of threshold value, by considering background signal, susceptibility and signal intensity or device
Error range is utilized from the individual threshold to signal at a temperature of 60 DEG C of the selection of above-mentioned pipe.In the present embodiment, 60 are used for
The individual threshold of signal at a temperature of DEG C is confirmed as " 200 ".
As shown in Fig. 1 D and Fig. 1 E, according to the individual threshold at a temperature of 60 DEG C, confirmation is above-mentioned in pipe (2) and pipe (3)
The presence of gonococcus, in particular, CtValue obtains 31.32 and 35.83 respectively.It can not find out in pipe (1) and pipe (4) to gonococcus
CtValue.
This result is expressed as follows: the detection comprising the signal detection at 2 temperature and labeled oligonucleotide cutting and
Extend in real-time method, (i) signal detection at a temperature of above-mentioned relatively high detection is detectable has above-mentioned relatively high detection
The target nucleic acid sequence of temperature, the signal that (ii) is obtained at a temperature of above-mentioned relatively high detection temperature and above-mentioned relatively low detection
It can be used in the target nucleic acid sequence that detection has above-mentioned relatively low detection temperature.
Therefore, the detection of available signal detection comprising 1 sense channel and at a temperature of different and label few nucleosides
Acid cutting and extension real-time polymerase chain reaction, detect 2 target nucleic acids in 1 reaction vessel.
Embodiment 2: based on include different temperatures under signal detection TaqMan/ detection and labeled oligonucleotide cutting and
Extend 2 targets detection of real-time polymerase chain reaction
The present inventor, which utilizes, to be cut comprising the TaqMan/ detection under 1 sense channel and different temperatures with labeled oligonucleotide
It cuts and extends real-time polymerase chain reaction and carried out the experiment that could detect two target nucleic acids in 1 reaction vessel.
For the extension of upstream primer and downstream primer, the cutting of TaqMan probe and detection and labeled oligonucleotide segment
Cutting and detection and labeled oligonucleotide segment extension, and used the Taq deoxyribose core with 5 ' nucleases
Sour polymerase.Using the genomic deoxyribonucleic acid of the genomic deoxyribonucleic acid of gonococcus and Chlamydia as target nucleus
Acid sequence uses.
In order to detect Chlamydia, and TaqMan real-time polymerase chain reaction is used.Chlamydia if it exists,
Then TaqMan probe is released the segment of label by cutting.It can be expanded by measuring the signal from the segment of above-mentioned label
Curve.In order to detect gonococcus, and detection and labeled oligonucleotide is used to cut and extend real-time polymerase chain reaction side
Method.
In the present embodiment, it is contemplated that signal generating mechanism selects " 72 DEG C " to examine as the signal for Chlamydia
Testing temperature selects " 60 DEG C " as the signal detection temperature for being used for gonococcus.According to prolonging there are generated for above-mentioned gonococcus
Stretching dimer has the adjustable Tm value adjusted based on its sequence and length.In the present embodiment, above-mentioned extension dimerization
The sequence and length of object be designed to 60 DEG C at a temperature of form above-mentioned dimer signal be provided, but in 72 DEG C of temperature
Under do not form above-mentioned dimer and be dissociated, to provide different signals.At a temperature of 72 DEG C of detection, generate for above-mentioned
The signal of Chlamydia simultaneously detects signal.At a temperature of 60 DEG C of detection, not only generate for above-mentioned Chlamydia
Signal, but also generate the signal for above-mentioned gonococcus and be detected.
TaqMan probe its 5 '-end by fluorescent reporter label (CAL Fluor Red610) and its
3 '-ends mark (sequence 9) by quenching molecules (BHQ-2).Above-mentioned detection and labeled oligonucleotide and capture and templating widow core
The 3 ' of thuja acid-end is blocked in a manner of preventing it from extending by carbon spacer region.Above-mentioned capture and templating oligonucleotides its 5 '-
End at quenching molecules (BHQ-2) by marking and at its templating position by fluorescent reporter (CAL Fluor Red
610) (sequence 4) is marked.
Prepare to separately include Chlamydia, gonococcus, Chlamydia and the mixture of gonococcus and pair without target
According to 4 reaction tubes of group.
The upstream primer, downstream primer, detection and the labeled oligonucleotide that use in the present embodiment and capture and templating
The sequence of oligonucleotides is as follows.
NG_F 5 '-TACGCCTGCTACTTTCACGCTIIIIIGTAATCAGATG-3 ' (sequence 1)
NG_R 5 '-CAATGGATCGGTATCACTCGCIIIIICGAGCAAGAAC-3 ' (sequence 2)
NG_PTO 5’-GTACGCGATACGGGCCCCTCATTGGCGTGTTTCG [C3 spacer] -3 ' (sequence 3)
NG_CTO 5’-[BHQ-2]TTTTTTTTTTTTTTTTTTTG[T(Cal Fluor Red 610)]
ACTGCCCGTATCGCGTAC [C3 spacer] -3 ' (sequence 4)
CT_F 5 '-GAGTTTTAAAATGGGAAATTCTGGTIIIIITTTGTATAAC-3 ' (sequence 5)
CT_R 5 '-CCAATTGTAATAGAAGCATTGGTTGIIIIITTATTGGAGA-3 ' (sequence 6)
CT_P 5 '-[Cal Fluor Red610] CATCAGAAGCTGTCATTTTGGCTGCG [BHQ-2] -3 ' (sequence 9)
(I: deoxyinosine)
(the 5 '-label positions that underlined text indicates detection and labeled oligonucleotide)
Using including the target nucleic acid (base of the chlamydia trachomatis of the genomic deoxyribonucleic acid and 10pg of the gonococcus of 10pg
Because of the gene of the chlamydia trachomatis of the genomic deoxyribonucleic acid and 10pg of group DNA or the gonococcus of 10pg
Group DNA mixture), 10pmole for gonococcus target amplification upstream primer (sequence 1) and 10pmole
Downstream primer (sequence 2), 5pmole detection and labeled oligonucleotide (sequence 3), the capture of 1pmole and templating widow's core
Thuja acid (sequence 4), the upstream primer (sequence 5) that expands of the target for chlamydia trachomatis of 10pmole and the downstream of 12pmole are drawn
(final, 200 μM of triphosphoric acid is de- for 2 × main mixed liquor of object (sequence 6), the TaqMan probe of 1pmole (sequence 9) and 10 μ l
The MgCl of oxygen nucleotide (dNTPs), 2mM2And the Taq deoxyribonucleic acid polymerase of 2U) 20 μ l final volumes carried out reality
When polymerase chain reaction.The pipe containing above-mentioned reaction mixture is set to be located at real time thermocycler (CFX96, Bole company Bio-
Rad), and 50 DEG C at a temperature of react after five minutes, 95 DEG C at a temperature of be denaturalized 15 minutes, and in 95 DEG C of temperature
Lower denaturation 30 seconds, 60 DEG C at a temperature of denaturation 60 seconds, implement repeatedly in 72 DEG C of at a temperature of 30 seconds modes of denaturation
50 times.60 DEG C of each circulation and 72 DEG C at a temperature of carried out the detection of signal.
As shown in Figure 2 A, in the presence of chlamydia trachomatis (pipe (1) and pipe (3)), 72 DEG C at a temperature of detect
Signal is gone out.There is only gonococcus (pipe (2)), 60 DEG C at a temperature of detected signal, but at 72 DEG C
At a temperature of do not detect signal.In the case where target nucleic acid is not present, (pipe (4)) does not detect signal.The result of Fig. 2A is shown
Out, former for the trachoma clothing with relatively high detection temperature in 72 DEG C of the at a temperature of generation as relatively high detection temperature
The signal of body, but the signal to gonococcus is not generated.Therefore, following identification: using at a temperature of above-mentioned relatively high detection
Signal detection at least can determine the presence of the chlamydia trachomatis with above-mentioned relatively high detection temperature.And in pipe (2), lead to
Cross using because signal at a temperature of above-mentioned relatively high detection be not present and relatively low detection at a temperature of above-mentioned signal
The difference in the presence of caused by, it may be determined that the presence of the gonococcus with above-mentioned relatively low detection temperature.
In order to test from have it is above-mentioned it is relatively high detection temperature chlamydia trachomatis signal there are the case where or not
In the presence of whether could also detect the presence of the gonococcus with above-mentioned relatively low detection temperature, and connect using a variety of
Nearly method calculates between above-mentioned relatively high detection temperature and the signal detected at a temperature of above-mentioned relatively low detection
Difference (Fig. 2 B~2E).
Above-mentioned Fig. 2 B show the RFU value of the endpoint at a temperature of 72 DEG C and 60 DEG C ratio (all RFU values derive from " base
Cableties remove curve matching (baseline subtracted curve fit) " data are analyzed to send out).It is former that there is only trachoma clothing
The ratio of (pipe (3)) is 1.8 in the presence of the ratio of (pipe (1)) is 1.1 when body, but Chlamydia and gonococcus are equal.Also,
The ratio of (pipe (4)) is difference 1278.0 and 1.0 when there is only the ratio of (pipe (2)) when gonococcus and without any target nucleic acid.For
The existence or non-existence of determining gonococcus, and applicable threshold 1.2.According to above-mentioned threshold value, the confirmation leaching in pipe (2) and pipe (3)
The presence of coccus, and confirm in pipe (1) and pipe (4) without gonococcus.Consider from the final-ratio for only including Chlamydia
(End-Raio) above-mentioned threshold value is determined.With selecting a property, as managed (2), it is possible to establish that for relatively high detection temperature
Chlamydia be not present in the case where, instead of threshold value 1.2, can independently set for above-mentioned at a temperature of relatively low detection
Signal.
Using 72 DEG C and 60 DEG C at a temperature of signal other close in method, in each circulation calculate 72 DEG C and 60
The ratio of fluorescence signal at a temperature of DEG C, and to above-mentioned ratio (all RFU values source handled to draw of circulation drawing
In no baseline deduction (no baseline subtraction) " data are analyzed to send out).In order to determine gonococcus presence or
There is no used threshold value 0.1.Above-mentioned threshold value is determined in a manner of considering that the ratio from the pipe for only including Chlamydia is drawn.
As shown in Figure 2 C, the presence of gonococcus, C are confirmed in pipe (2) and pipe (3)tValue is respectively 37.88 and 37.20.In pipe (1) and
C is not obtained in pipe (4)tValue.In each cycle, glimmering at a temperature of deducting 60 DEG C in the fluorescence intensity at a temperature of 72 DEG C
Luminous intensity, thus instead of the mode of calculating ratio, and in order to detect target, needle can draw to the above results for circulation.
It detects using the signal of Chlamydia is indicated at a temperature of relatively high detection with above-mentioned relatively low
In the existing another method for detecting the gonococcus of temperature, in order to analyze the at a temperature of letter that obtains at 60 DEG C in each pipe
Number, and it is applicable in individual threshold.In the case where detecting to indicate the existing signal of chlamydia trachomatis at a temperature of 72 DEG C, pass through
Each End-RFU at a temperature of 72 DEG C to calculate from above-mentioned pipe for the signal at a temperature of 60 DEG C multiplied by threshold value (1.2)
Individual threshold.Final-ratio (referring to Fig. 2 B) from the pipe for only including Chlamydia is considered to determine above-mentioned threshold value (1.2).
In the case where not detecting signal at a temperature of 72 DEG C, according to the common setting method of threshold value, by consider background signal,
The error range of susceptibility and signal intensity or device, from above-mentioned pipe selection 60 DEG C at a temperature of the individual threshold to signal
To utilize.In the present embodiment, the individual threshold for the signal at a temperature of 60 DEG C is confirmed as " 200 ".
As shown in Fig. 2 D and Fig. 2 E, according to the individual threshold at a temperature of 60 DEG C, confirmation is above-mentioned in pipe (2) and pipe (3)
The presence of gonococcus, in particular, CtValue obtains 36.21 and 37.07 respectively.It can not find out in pipe (1) and pipe (4) to gonococcus
CtValue.
This result is expressed as follows: in the TaqMan/ detection comprising the signal detection at 2 temperature and labeled oligonucleotide
In cutting and extension real-time method, (i) signal detection at a temperature of above-mentioned relatively high detection is detectable with above-mentioned opposite
Compared with the target nucleic acid sequence of the Supreme People's Procuratorate's testing temperature, (ii) is obtained at a temperature of above-mentioned relatively high detection temperature and above-mentioned relatively low detection
The signal obtained can be used in the target nucleic acid sequence that detection has above-mentioned relatively low detection temperature.
Therefore, using the TaqMan/ detection of the signal detection at a temperature of including 1 sense channel and being different and mark
Remember oligonucleotides cutting and extend real-time polymerase chain reaction, 2 target nucleic acids are detected in 1 reaction vessel.
Embodiment 3: two targets detection based on real-time polymerase chain reaction and melting analysis
The present inventor has been carried out using the combination in 1 sense channel and real-time polymerase chain reaction and melting analysis
The experiment of two target nucleic acids could be detected in 1 reaction vessel.
For cutting for the extension of upstream primer and downstream primer, the cutting of TaqMan probe, detection and labeled oligonucleotide
It cuts and detects and the extension of labeled oligonucleotide segment, and used the Taq DNA with 5 ' nucleases poly-
Synthase.Using the genomic deoxyribonucleic acid of the genomic deoxyribonucleic acid of gonococcus and Chlamydia as target nucleic acid sequence
Column use.
In order to detect Chlamydia, and TaqMan real-time polymerase chain reaction is used, made to detect gonococcus
With detection and labeled oligonucleotide cutting and extend melting analysis.Chlamydia if it exists, then TaqMan probe is cut
And release the segment of label.Gonococcus if it exists, then detection and labeled oligonucleotide are cut and are generated detection and mark few core
Acid fragments.Above-mentioned detection and labeled oligonucleotide segment are annealed at the capture position of capture and templating oligonucleotides, In
It captures and extends on the templating position with templating oligonucleotides, and form capture and templating oligonucleotides and extend dimer
(dimer captures and templating oligonucleotides).
During real-time polymerase chain reaction, in order to which the fluorescence that the cutting only detected by TaqMan probe generates is believed
Number, and dimer can not be formed since extension dimer is dissociated, thus cut from by detection and labeled oligonucleotide
The above-mentioned extension dimer cut and extended to form carries out above-mentioned fluorescence signal detection at a temperature of not generating signal.In order in unwinding
It obtains in the process and the existing unwinding peak of dimer is extended according to the expression that the presence of gonococcus is formed, and determine signal.
TaqMan probe its 5 '-end by fluorescent reporter (CAL Fluor Red 610) mark and its 3 '-
End marks (sequence 9) by quenching molecules (BHQ-2).Above-mentioned detection and labeled oligonucleotide and capture and templating oligonucleotides
Its 3 '-ends blocked in a manner of preventing it from extending by carbon spacer region.Above-mentioned capture and templating oligonucleotides its 5 '-
End is marked by quenching molecules (BHQ-2) and at its templating position by fluorescent reporter (CAL Fluor Red 610)
It marks (sequence 4).
The upstream primer, downstream primer, detection and the labeled oligonucleotide that use in the present embodiment and capture, templating widow
The sequence of nucleotide and TaqMan probe is as follows.
NG_F 5 '-TACGCCTGCTACTTTCACGCTIIIIIGTAATCAGATG-3 ' (sequence 1)
NG_R 5 '-CAATGGATCGGTATCACTCGCIIIIICGAGCAAGAAC-3 ' (sequence 2)
NG_PTO 5’-GTACGCGATACGGGCCCCTCATTGGCGTGTTTCG [C3 spacer] -3 ' (sequence 3)
NG_CTO 5’-[BHQ-2]TTTTTTTTTTTTTTTTTTTG[T(Cal Fluor Red 610)]
ACTGCCCGTATCGCGTAC [C3 spacer] -3 ' (sequence 4)
CT_F 5 '-GAGTTTTAAAATGGGAAATTCTGGTIIIIITTTGTATAAC-3 ' (sequence 5)
CT_R 5 '-CCAATTGTAATAGAAGCATTGGTTGIIIIITTATTGGAGA-3 ' (sequence 6)
CT_P 5 '-[Cal Fluor Red610] CATCAGAAGCTGTCATTTTGGCTGCG [BHQ-2] -3 ' (sequence 9)
(I: deoxyinosine)
(the 5 '-label positions that underlined text indicates detection and labeled oligonucleotide)
Using including the target nucleic acid (base of the chlamydia trachomatis of the genomic deoxyribonucleic acid and 10pg of the gonococcus of 10pg
Because of the gene of the chlamydia trachomatis of the genomic deoxyribonucleic acid and 10pg of group DNA or the gonococcus of 10pg
Group DNA mixture), 10pmole for gonococcus target amplification upstream primer (sequence 1) and 10pmole
Downstream primer (sequence 2), 5pmole detection and labeled oligonucleotide (sequence 3), the capture of 1pmole and templating widow's core
Thuja acid (sequence 4), the upstream primer (sequence 5) that expands of the target for chlamydia trachomatis of 10pmole and the downstream of 12pmole are drawn
(final, 200 μM of triphosphoric acid is de- for 2 × main mixed liquor of object (sequence 6), the TaqMan probe of 1pmole (sequence 9) and 10 μ l
The MgCl of oxygen nucleotide, 2mM2And the Taq deoxyribonucleic acid polymerase of 2U) 20 μ l final volumes carried out real time aggregation enzyme
Chain reaction.The pipe containing above-mentioned reaction mixture is set to be located at real time thermocycler (CFX96, Bole company), and at 50 DEG C
At a temperature of react after five minutes, 95 DEG C at a temperature of denaturation 15 minutes and with 95 DEG C at a temperature of denaturation 30 seconds, 60
It is denaturalized 60 seconds at a temperature of DEG C, implements repeatedly 50 times in 72 DEG C of at a temperature of 30 seconds modes of denaturation.In each circulation
The detection of signal has been carried out at a temperature of 72 DEG C.After above-mentioned reaction 55 DEG C at a temperature of make above-mentioned tube reaction 5 minutes, from 55
Fluorescence signal is measured during DEG C improving temperature to 95 DEG C with 0.5 DEG C of interval to have carried out curve analysis.
As shown in figure 3, in the presence of CT (pipe (1)), although being obtained during real-time polymerase chain reaction
Amplification curve, but do not observe unwinding peak in melting analysis.In the presence of gonococcus (pipe (2)), although in reality
When polymerase chain reaction during do not obtain amplification curve, but observed in melting analysis with estimated Tm value (66 DEG C)
The presence according to gonococcus and the unwinding peak that is formed extends the unwinding peak of dimer.In the presence of CT and gonococcus
(pipe (3)), real-time polymerase chain reaction process and melting analysis observe signal in the process.In the feelings that target nucleic acid is not present
(pipe (4)) does not observe any signal under condition.
This result indicates that 1 sense channel is used only by the combination of real-time polymerase chain reaction and melting analysis
It can detect multiple target nucleic acids.
Embodiment 4: the nucleotide based on the real-time polymerase chain reaction comprising signal detection at different temperatures is more
State property genotyping
The present inventor could be suitable at 1 the real-time polymerase chain reaction comprising the signal detection under different temperatures
It is tested in reaction vessel using the nucleotide polymorphisms genotyping of 1 sense channel.
For the cutting and detection of the extension of upstream primer and downstream primer, detection and labeled oligonucleotide and the few core of label
The extension of acid fragments, and used the Taq deoxyribonucleic acid polymerase with 5 ' nucleases.By MTHFR (C677T)
Wild type (C) homozygote, saltant type (T) homozygote and the heterozygote of human genome DNA are as target nucleic acid sequence
It uses.
In order to detect wild type (C) allele and saltant type of MTHFR (C677T) human genome DNA
(T) allele, and detection and labeled oligonucleotide has been used to cut and extend real-time polymerase chain reaction.If target equipotential base
Because existing, then detection and labeled oligonucleotide are cut, and generate detection and labeled oligonucleotide segment.Above-mentioned detection and label
Oligonucleotide fragment is annealed at the capture position of capture and templating oligonucleotides, is capturing the mould with templating oligonucleotides
Extend on plate position and forms capture and templating oligonucleotides and extend dimer (dimer capture and templating few nucleosides
Acid).Signal is provided by the formation of above-mentioned extension dimer, signal is measured in the case where extending dimer-formation temperature, so as to obtain
Obtain amplification curve.
In the present embodiment, it is contemplated that signal generating mechanism selects " 72 DEG C " as wild type (C) allele
Signal detection temperature selects " 55 DEG C " as the signal detection temperature for being used for saltant type (T) allele.According to above-mentioned wild type
(C) allele or having there are extension dimer generated for saltant type (T) allele are obtained based on its sequence and length
To adjustable Tm value of adjusting.In the present embodiment, for the sequence of the extension dimer of above-mentioned wild type (C) allele
And length be designed to 72 DEG C at a temperature of form above-mentioned dimer signal be provided.On the other hand, for above-mentioned saltant type
(T) sequence of the extension dimer of allele and length be designed to 55 DEG C at a temperature of form above-mentioned dimer and provide
Signal, but 72 DEG C at a temperature of do not form above-mentioned dimer and be dissociated, to not provide signal.In 72 DEG C of detection temperature
Under degree, generates the signal to above-mentioned wild type (C) allele and detect signal.At a temperature of 55 DEG C of detection, not only occur
For the signal of above-mentioned wild type (C) allele, and the signal for being directed to above-mentioned saltant type (T) allele is generated, and examine
It measures.
Its 3 '-ends of above-mentioned detection and labeled oligonucleotide and capture and templating oligonucleotides are to prevent prolonging for its
The mode stretched is blocked by carbon spacer region.Above-mentioned capture and templating oligonucleotides are marked in its 5 '-end by quenching molecules (BHQ-2)
Remember and (sequence 13 and sequence 15) is marked by fluorescent reporter (CAL Fluor Red 610) at its templating position.
The upstream primer, downstream primer, detection and the labeled oligonucleotide that use in the present embodiment and capture and templating
The sequence of oligonucleotides is as follows.
M677_F 5 '-CCACCCCGAAGCAGGGAIIIIIGAGGCTGACC-3 ' (sequence 10)
M677_R 5 '-CAAGTGATGCCCATGTCGGIIIIIGCCTTCACAA-3 ' (sequence 11)
M677_W_PTO 5’-GGTCCCGACGTTAGCTCCCGCAGACACCTTCTCCTTC [C3 spacer] -3 ' (sequences
Column 12)
M677_W_CTO 5’-[BHQ-2]CCTCGGTGCCACGCCATCGG[T(Cal Fluor Red 610)]
TCTTCTAACGTCGGGACC [C3 spacer] -3 ' (sequence 13)
M677_M_PTO 5’-ACGTCGATTCGCACTCCCGCAGACACCTTCTCCTTCAA [C3 spacer] -3 ' (sequences
Column 14)
M677_M_CTO 5’-[BHQ-2]TTTTTTTTTTTTTTTTTTTT[T(Cal Fluor Red 610)]
ATTCTGCGAATCGACGT [C3 spacer] -3 ' (sequence 15)
(I: deoxyinosine)
(the 5 '-label positions that underlined text indicates detection and labeled oligonucleotide)
Using comprising target nucleic acid, (wild type (C) of MTHFR (C677T) human genome DNA of 1ng is homozygous
Saltant type (T) homozygote of MTHFR (C677T) human genome DNA of son, 1ng or the MTHFR of 1ng
(C677T) heterozygote of human genome DNA), the upstream primer (sequence 10) of 5pmole, 5pmole downstream
The capture of primer (sequence 11), the respectively detection of 3pmole and labeled oligonucleotide (sequence 12 and sequence 14), difference 1pmole
With 2 × main mixed liquor (final, 200 μM of triphosphoric acid deoxidation core of templating oligonucleotides (sequence 13 and sequence 15) and 10 μ l
The MgCl of thuja acid, 2mM2And the Taq deoxyribonucleic acid polymerase of 2U) the final volume of 20 μ l carried out real time aggregation enzyme chain
Formula reaction.The pipe containing above-mentioned reaction mixture is set to be located at real time thermocycler (CFX96, Bole company), and in 50 DEG C of temperature
The lower reaction of degree after five minutes, 95 DEG C at a temperature of be denaturalized 15 minutes, and with 95 DEG C at a temperature of denaturation 30 seconds, 60
It is denaturalized 60 seconds at a temperature of DEG C, implements repeatedly 50 times in 72 DEG C of at a temperature of 30 seconds modes of denaturation.In each circulation
The detection of signal has been carried out at a temperature of 60 DEG C and 72 DEG C.
As shown in Figure 4 A, in the presence of wild type (C) homozygote (pipe (1)) or in the presence of heterozygote
(pipe (3)), 72 DEG C and 55 DEG C at a temperature of detected signal.(pipe in the presence of saltant type (T) homozygote
(2)), 55 DEG C at a temperature of detected strong signal, but 72 DEG C at a temperature of detect faint signal.Target not
In the presence of (pipe (4)) do not detect any signal.
Above-mentioned Fig. 4 B show the RFU value of the endpoint at a temperature of 72 DEG C and 60 DEG C ratio (whole RFU values derive from " base
Cableties are sent out except curve fitting analysis data).The ratio of (pipe (1)) is 1.2 in the presence of wild type (C) homozygote, still
The ratio of (pipe (3)) is 1.9 in the presence of heterozygote.Difference expression between this above-mentioned 2 ratios is including heterozygote
Saltant type (T) allele exists together with wild type (C) allele in pipe.On the other hand, homozygous in saltant type (T)
In the case of (pipe (2)), due to 72 DEG C at a temperature of RFU value it is low thus opposite from the ratio of saltant type (T) homozygote acquisition
It is very high.If consider in heterozygote wild-type allele and mutant allele with the ratio of 1:1 there are the fact,
In the presence of mutant homozygote, it is known that in above-mentioned 72 DEG C of the at a temperature of faint signal that detects be false positive letter
Number.According to the method for the invention for nucleotide polymorphisms genotyping, it may be determined that whether above-mentioned rate value is in above-mentioned phase
To compared with the error signal at a temperature of high detection.
This result indicates that the real-time polymerase chain reaction comprising signal detection is applicable to utilize 1 at different temperatures
The nucleotide polymorphisms genotyping of a sense channel, at a temperature of above-mentioned relatively high detection temperature and relatively low detection
Above-mentioned signal obtain difference can be used for nucleotide polymorphisms genotyping.
Embodiment 5: based on comprising the signal detection under different temperatures TaqMan/ detection and labeled oligonucleotide cutting and
Extend multiple targets detection of real-time polymerase chain reaction
The present inventor utilizes the TaqMan/ detection comprising the signal detection under 1 sense channel and different temperatures and label
Oligonucleotides cutting and extension real-time polymerase chain reaction have carried out that three target nucleic acids could be detected in a reaction vessel
Experiment.
For cutting for the extension of upstream primer and downstream primer, the cutting of TaqMan probe, detection and labeled oligonucleotide
It cuts and detects and the extension of labeled oligonucleotide segment, and used the Taq DNA with 5 ' nucleases poly-
Synthase.By the genomic deoxyribonucleic acid of gonococcus, the genomic deoxyribonucleic acid and mycoplasma genitalium of Chlamydia
Genomic deoxyribonucleic acid used as target nucleic acid sequence.
In order to detect mycoplasma genitalium, and TaqMan real-time polymerase chain reaction is used.It is former in order to detect sand holes clothing
Body and gonococcus, and detection and labeled oligonucleotide has been used to cut and extend real-time polymerase chain reaction.
In the present embodiment, it is contemplated that signal generating mechanism selects " 95 DEG C " to examine as the signal for mycoplasma genitalium
Testing temperature selects " 72 DEG C " as the signal detection temperature for being used for Chlamydia, selects " 72 DEG C " as gonococcus
Signal detection temperature.In the present embodiment, the sequence of the extension dimer of above-mentioned Chlamydia or gonococcus and length are set
Count into 72 DEG C or 60 DEG C at a temperature of form above-mentioned dimer signal be provided, but 95 DEG C at a temperature of do not formed it is above-mentioned
Dimer is simultaneously dissociated, to not provide signal.At a temperature of 72 DEG C of detection, not only generate for above-mentioned mycoplasma genitalium
Signal, but also generate the signal for above-mentioned CT and be detected.Also, at a temperature of 60 DEG C of detection, not only generates and be directed to
The signal of above-mentioned mycoplasma genitalium and CT, but also generate the signal for above-mentioned NG and be detected.
TaqMan probe its 5 '-end by fluorescent reporter (CAL Fluor Red 610) mark and its 3 '-
End marks (sequence 18) by quenching molecules (BHQ-2).Above-mentioned detection and labeled oligonucleotide and capture and templating few nucleosides
Acid is blocked in a manner of preventing its extension by carbon spacer region.Above-mentioned capture and templating oligonucleotides are in its 5 '-end by sudden
Go out molecule (BHQ-2) label and its templating position by fluorescent reporter (CAL Fluor Red610) mark (sequence 4
And sequence 8).
The mixing of gonococcus, chlamydia trachomatis body, mycoplasma genitalium, gonococcus and Chlamydia is prepared to separately include
Object, the mixture of gonococcus and mycoplasma genitalium, chlamydia trachomatis and the mixture of mycoplasma genitalium, gonococcus, sand holes clothing original
8 reaction tubes of the mixture and the control group without target of body and reproduction Chlamydia.
Upstream primer, downstream primer, detection and labeled oligonucleotide, capture and the templating used in the present embodiment is few
The sequence of nucleotide and TaqMan probe is as follows.
NG_F 5 '-TACGCCTGCTACTTTCACGCTIIIIIGTAATCAGATG-3 ' (sequence 1)
NG_R 5 '-CAATGGATCGGTATCACTCGCIIIIICGAGCAAGAAC-3 ' (sequence 2)
NG_PTO 5’-GTACGCGATACGGGCCCCTCATTGGCGTGTTTCG [C3 spacer] -3 ' (sequence 3)
NG_CTO 5’-[BHQ-2]TTTTTTTTTTTTTTTTTTTG[T(Cal Fluor Red 610)]
ACTGCCCGTATCGCGTAC [C3 spacer] -3 ' (sequence 4)
CT_F 5 '-GAGTTTTAAAATGGGAAATTCTGGTIIIIITTTGTATAAC-3 ' (sequence 5)
CT_R 5 '-CCAATTGTAATAGAAGCATTGGTTGIIIIITTATTGGAGA-3 ' (sequence 6)
CT_PTO 5’-GATTACGCGACCGCATCAGAAGCTGTCATTTTGGCTGCG [C3 spacer] -3 ' (sequences
7)
CT_CTO 5’-[BHQ-2]GCGCTGGATACCCTGGACGA[T(Cal Fluor Red 610)]
ATGTGCGGTCGCGTAATC [C3 spacer] -3 ' (sequence 8)
MG-F 5 '-AAAACCCACGGAAATGATGAGAIIIIIATTGGTTCTAC-3 ' (sequence 16)
MG-R 5 '-CTCGTTAATTTACCTATTCCATTTTGIIIIICTGATAAAAG-3 ' (sequence 17)
- 3 ' (sequence of MG-P 5 '-[CAL Fluor Red610] GAGTTCTTTCAAGAACAGCAAGAGGTGT [BHQ-2]
18)
(I: deoxyinosine)
(the 5 '-label positions that underlined text indicates detection and labeled oligonucleotide)
Using comprising target nucleic acid (genomic deoxyribonucleic acid of the gonococcus of 10pg, the chlamydia trachomatis of 10pg base
Because of a group DNA, distinguishes the gonococcus of 10pg and the genomic deoxyribonucleic acid of chlamydia trachomatis, distinguishes 10pg's
The mixture of the genomic deoxyribonucleic acid of gonococcus and mycoplasma genitalium, the respectively chlamydia trachomatis of 10pg and genital branch are former
The mixture of the genomic deoxyribonucleic acid of body, the respectively gonococcus, chlamydia trachomatis of 10pg and the gene of mycoplasma genitalium
The mixture of group DNA), the upstream primer (sequence 1) and 5pmole of the target amplification for gonococcus of 5pmole
Swim primer (sequence 2), the detection of 3pmole and labeled oligonucleotide (sequence 3), the capture of 1pmole and templating oligonucleotides
Downstream primer (the sequence of upstream primer (sequence 5) and 5pmole that the target for chlamydia trachomatis of (sequence 4), 5pmole expand
Column 7), the detection of 3pmole and labeled oligonucleotide (sequence 7), the capture of 1pmole and templating oligonucleotides (sequence 8),
The downstream primer (sequence 17) of upstream primer (sequence 16) and 5pmole that the target for mycoplasma genitalium of 5pmole expands,
The TaqMan probe (sequence 18) of 1pmole and 10 μ l 2 × main mixed liquor (it is final, 200 μM of triphosphate deoxy-nucleotide,
The MgCl of 2mM2And the Taq deoxyribonucleic acid polymerase of 2U) 20 μ l final volumes carried out real-time polymerase chain reaction.
Be located at the pipe containing above-mentioned reaction mixture real time thermocycler (CFX96, Bole company), and 50 DEG C at a temperature of react
After five minutes, 95 DEG C at a temperature of be denaturalized 15 minutes, and in 95 DEG C of at a temperature of denaturation 30 seconds and in 60 DEG C of temperature
The lower denaturation of degree 60 seconds, 72 DEG C at a temperature of denaturation 30 seconds mode implement repeatedly 50 times.At 60 DEG C of each circulation and
The detection of signal has been carried out at a temperature of 72 DEG C and 95 DEG C.
As shown in Fig. 5 A, Fig. 5 B and Fig. 5 C, pipe (3), pipe (5), pipe (6) and pipe (7) in, 95 DEG C at a temperature of detect
Signal out exists in which at least can determine the mycoplasma genitalium with opposite highest detection temperature (95 DEG C).
Using because the signal under above-mentioned opposite highest detection temperature (95 DEG C) be not present and above-mentioned opposite intermediate detection temperature
Spend (relatively middle detection temperature；72 DEG C) under signal presence caused by difference can be true
It is scheduled on pipe (2) and manages the presence of the chlamydia trachomatis with above-mentioned opposite intermediate detection temperature (72 DEG C) in (4).
In particular, using because the signal under above-mentioned opposite intermediate detection temperature (72 DEG C) be not present and above-mentioned relatively minimum inspection
Difference caused by the presence of signal under testing temperature (60 DEG C) can determine has above-mentioned opposite lowest detection temperature in pipe (1)
The presence of the gonococcus of (60 DEG C).
Existing chlamydia trachomatis or gonococcus together with other targets could be confirmed in a reaction vessel in order to investigate,
And by the subtraction of End-RFUs (End- △ RFU) above-mentioned 95 DEG C, 72 DEG C and 60 DEG C at a temperature of the signal that detects it
Between difference.
Fig. 5 D indicate 95 DEG C and 72 DEG C at a temperature of using endpoint RFU value calculate End- △ RFUs (whole RFU values
" baseline deduction curve matching (baseline subtracted curve fit) " in the software of device analyzes data
To send out).In order to determine the presence of chlamydia trachomatis and applicable threshold " 300 ".Consider from the pipe (pipe for not including Chlamydia
(1), (3) are managed and manage (5)) End- △ RFUs determine above-mentioned threshold value.According to above-mentioned threshold value determined pipe (2), pipe (4),
It manages (6) and manages the presence of chlamydia trachomatis in (7).
Fig. 5 E indicate 72 DEG C and 60 DEG C at a temperature of using endpoint RFU value calculate End- △ RFUs (whole RFU values
Number is analyzed from " baseline deduction curve matching (the baseline subtracted curve fit) " in the software of device
According to sending out).In order to gonococcus there are applicable threshold " 800 ".Consider from do not include gonococcus pipe (pipe (2), pipe (3) and
Pipe (6)) End- △ RFUs determine above-mentioned threshold value.It has been determined according to above-mentioned threshold value in pipe (1), pipe (4), pipe (5) and pipe (7)
The presence of middle gonococcus.
This result is expressed as follows: in the TaqMan/ detection comprising the signal detection at 3 temperature and labeled oligonucleotide
In cutting and extension real-time method, (i) signal detection at a temperature of above-mentioned relatively high detection is detectable with above-mentioned opposite
Compared with the target nucleic acid sequence of the Supreme People's Procuratorate's testing temperature, (ii) has the target nucleus of the detection temperature lower than above-mentioned highest detection temperature in order to confirm
The difference obtained from signal at a temperature of different above-mentioned detections can be used in acid sequence.
Therefore, the TaqMan/ detection of available signal detection comprising a sense channel and at a temperature of different and mark
Remember oligonucleotides cutting and extend real-time polymerase chain reaction, to detect multiple target nucleic acids in 1 reaction vessel.
The present invention relates to following implementation:
1. a kind of method using 2 target nucleic acid sequences in different detection temperature detection samples characterized by comprising
Step (a), in 1 reaction vessel together with signal-generation mechanism for detecting above-mentioned 2 target nucleic acid sequences
Said sample is cultivated, and detects generated signal using the detector of single type, is examined by corresponding signal-generation mechanism
Each above-mentioned target nucleic acid sequence is surveyed, one in above-mentioned 2 target nucleic acid sequences has by corresponding above-mentioned signal-generation mechanism
Determining relatively high detection temperature, another is relatively low with determining by corresponding above-mentioned signal-generation mechanism
Temperature is detected, above-mentioned relatively high detection temperature is that can generate for the target nucleic acid sequence with above-mentioned relatively high detection temperature
The temperature of the signal of column, above-mentioned relatively low detection temperature are that can generate for the target with above-mentioned relatively low detection temperature
The temperature of the signal of nucleic acid sequence and the signal for the target nucleic acid sequence with above-mentioned relatively high detection temperature, by above-mentioned
The signal that 2 signals-generation mechanism generates is not distinguished by the detector of above-mentioned single type, in above-mentioned relatively high detection temperature
And above-mentioned detection is carried out at a temperature of above-mentioned relatively low detection；And
Step (b) determines the presence of 2 target nucleic acid sequences according to the signal detected in above-mentioned steps (a), (i) root
The target nucleic acid sequence with above-mentioned relatively high detection temperature is determined according to the signal detected at a temperature of above-mentioned relatively high detection
The presence of column, (ii) is according to the signal gone out in above-mentioned relatively high detection temperature detection and in above-mentioned relatively low detection temperature
Under difference between the signal that detects determine the presence with the target nucleic acid sequence of above-mentioned relatively low detection temperature.
2. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, carries out above-mentioned steps (a) during with the amplification of signal of nucleic acid amplification.
3. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, carries out above-mentioned steps (a) during the amplification of signal of free nucleic acid amplification.
4. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, signal-generation mechanism for each above-mentioned target nucleic acid sequence is to be generated in a manner of being formed dependent on dimer
Signal-generation mechanism of signal.
5. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, signal-generation mechanism for each above-mentioned target nucleic acid sequence is to utilize to depend on and above-mentioned target nucleic acid sequence
The mode of the cutting of the mediation oligonucleotides of specific hybrid forms signal-generation mechanism of dimer.
6. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, signal-generation mechanism for the target nucleic acid sequence with above-mentioned relatively high detection temperature is few based on detection
Signal-generation mechanism of the cutting of nucleotide, for signal-production of the target nucleic acid sequence with above-mentioned relatively low detection temperature
Life structure is the signal-generation mechanism formed based on dimer.
7. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, signal-generation mechanism for the target nucleic acid sequence with above-mentioned relatively high detection temperature is few based on detection
Signal-generation mechanism of the cutting of nucleotide, for signal-production of the target nucleic acid sequence with above-mentioned relatively low detection temperature
Life structure is to be formed by the way of dependent on the cutting with the mediation oligonucleotides of above-mentioned target nucleic acid sequence specific hybrid
The signal of dimer-generation mechanism signal-generation mechanism.
8. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, above-mentioned 2 signal-generation mechanisms include identical label, from the signal of above-mentioned label not by the inspection of single type
Survey device difference.
9. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, above-mentioned difference includes to the signal detected at a temperature of above-mentioned relatively high detection and above-mentioned relatively low
The difference that the signal detected at a temperature of detection carries out Mathematical treatment to obtain.
10. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, in the case where not detecting signal at a temperature of above-mentioned relatively high detection, it is contemplated that above-mentioned relatively high
Signal is not detected at a temperature of detection, determines that there is above-mentioned phase according to the signal detected at a temperature of above-mentioned relatively low detection
Presence to the target nucleic acid sequence of lower detection temperature.
11. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, in the presence of with the target nucleic acid sequence of above-mentioned relatively high detection temperature, above-mentioned using different
It detects in temperature detection sample in the method for 2 target nucleic acid sequences, in order to determine the target with above-mentioned relatively low detection temperature
The presence of nucleic acid sequence and utilize a reference value.
12. according to embodiment 11 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, the presence in order to determine the target nucleic acid sequence with above-mentioned relatively low detection temperature, above-mentioned using different
Detect temperature detection sample in 2 target nucleic acid sequences method in utilize a reference value, (i) with reacting in above-mentioned steps (a)
In other different reaction vessels of container and for detecting the signal-with the above-mentioned relatively high target nucleic acid sequence for detecting temperature
Generation mechanism cultivates the target nucleic acid sequence with above-mentioned relatively high detection temperature together, and (ii) is in above-mentioned relatively high detection temperature
After detecting signal at a temperature of degree and above-mentioned relatively low detection, (iii) is by finding out in above-mentioned relatively high detection temperature
Difference between the signal detected under degree and the signal detected at a temperature of above-mentioned relatively low detection is above-mentioned to obtain
A reference value.
13. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, above-mentioned 1 reaction vessel further includes at least one attachment device, above-mentioned at least one attachment device respectively includes using
In the 2 additional signal-generation mechanisms for detecting the target nucleic acid sequence other than above-mentioned 2 target nucleic acid sequences, in above-mentioned appearance
Device is mutually distinguishable the above-mentioned signal generated by each device of 2 signal-generation mechanisms, distinguishes by different types of detector
Detect above-mentioned signal.
14. according to embodiment 1 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, above-mentioned 2 target nucleic acid sequences include nucleotide diversity, one in above-mentioned 2 target nucleic acid sequences is comprising above-mentioned
One of nucleotide diversity type, another includes the another type in nucleotide diversity.
15. according to embodiment 14 using it is different detection temperature detection samples in 2 target nucleic acid sequences methods,
It is characterized in that, above-mentioned nucleotide diversity is single nucleotide polymorphism.
16. a kind of method of the nucleotide polymorphisms genotype using different detection temperature analysis sample nucleic acid sequences,
It is characterised by comprising:
Step (a), signal-generation mechanism in 1 reaction vessel and for detecting nucleotide polymorphisms allele
Sample of the culture comprising the nucleic acid sequence containing above-mentioned mononucleotide polymorphism site together, and utilize the detector of single type
Signal caused by detecting detects each above-mentioned nucleotide polymorphisms allele by corresponding signal-generation mechanism, above-mentioned
One in nucleotide polymorphisms allele has the relatively high detection temperature determined by corresponding signal-generation mechanism
Degree, another has the relatively low detection temperature determined by corresponding signal-generation mechanism, above-mentioned relatively high detection temperature
Degree is directed to the temperature of the signal with the above-mentioned relatively high nucleotide polymorphisms allele for detecting temperature for that can generate, on
Stating relatively low detection temperature is that can generate for the nucleotide polymorphisms equipotential base with above-mentioned relatively low detection temperature
The signal and needle set of cause have the temperature of the signal of the nucleotide polymorphisms allele of above-mentioned relatively high detection temperature, by upper
It states the signal that signal-generation mechanism generates not distinguished by the detector of above-mentioned single type, in above-mentioned relatively high detection temperature
And above-mentioned detection is carried out at a temperature of above-mentioned relatively low detection；And
Step (b), according in above-mentioned steps (a) the signal detected at a temperature of above-mentioned relatively high detection and
The difference between signal detected at a temperature of above-mentioned relatively low detection determines nucleotide polymorphisms genotype.
17. a kind of method using at least three target nucleic acid sequence in different detection temperature detection samples, which is characterized in that
Include:
Step (a), at least three signal-in 1 reaction vessel and for detecting above-mentioned at least three target nucleic acid sequence
Generation mechanism cultivates said sample together, and detects generated signal using the detector of single type, by corresponding letter
Number-generation mechanism detects above-mentioned at least three target nucleic acid sequence respectively, above-mentioned at least three target nucleic acid sequence is respectively provided with by phase
The different detection temperature that the above-mentioned signal answered-generation mechanism determines, above-mentioned detection temperature is that can not only generate with above-mentioned inspection
The target nucleic acid sequence of testing temperature and can also generate for higher than above-mentioned detection temperature detection temperature target nucleic acid sequence
Signal temperature, the signal generated by above-mentioned signal-generation mechanism is not distinguished by the detector of above-mentioned single type, each
Above-mentioned detection is carried out at a temperature of difference detection；And
Step (b) determines the presence of at least three target nucleic acid sequence according to the signal detected in above-mentioned steps (a), when
Determine the target nucleic acid sequence with the particular detection temperature in above-mentioned at least three target nucleic acid sequence there are when, according to being higher than
It the signal that is detected at a temperature of 1 or more detection of above-mentioned particular detection temperature and is detected at a temperature of above-mentioned particular detection
Difference between signal out determines the presence with the target nucleic acid sequence of above-mentioned particular detection temperature, in above-mentioned detection temperature
When above-mentioned particular detection temperature is opposite highest detection temperature, determined according to the signal detected at a temperature of above-mentioned particular detection
The presence of above-mentioned target nucleic acid sequence.
18. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that after determining the presence with the target nucleic acid sequence of opposite highest detection temperature first, to arrange in descending order
Successively determine that the existing mode with the target nucleic acid sequence of relatively low detection temperature carries out above-mentioned steps (b).
19. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that carry out above-mentioned steps (a) during with the amplification of signal of nucleic acid amplification.
20. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that carry out above-mentioned steps (a) during the amplification of signal of free nucleic acid amplification.
21. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that signal-generation mechanism for each above-mentioned target nucleic acid sequence is the side to be formed dependent on dimer
Signal-generation mechanism of formula generation signal.
22. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that signal-generation mechanism for each above-mentioned target nucleic acid sequence is to utilize to depend on and above-mentioned target nucleus
The mode of the cutting of the mediation oligonucleotides of acid sequence specific hybrid forms signal-generation mechanism of dimer.
23. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that signal-generation mechanism for the target nucleic acid sequence with above-mentioned opposite highest detection temperature be based on
Signal-the generation mechanism for detecting the cutting of oligonucleotides, signal-generation mechanism for remaining above-mentioned target nucleic acid sequence be based on
Signal-generation mechanism that dimer is formed.
24. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that signal-generation mechanism for the target nucleic acid sequence with above-mentioned opposite highest temperature detection temperature is base
In signal-generation mechanism of the cutting of detection oligonucleotides, signal-generation mechanism to remaining above-mentioned target nucleic acid sequence is to utilize
The signal-of dimer is formed in a manner of dependent on the cutting with the mediation oligonucleotides of above-mentioned target nucleic acid sequence specific hybrid
Generation mechanism.
25. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that above-mentioned at least three signal-generation mechanism includes identical label, from the signal of above-mentioned label not coverlet
The detector of one type is distinguished.
26. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that above-mentioned difference includes to detect at a temperature of being higher than 1 or more detection of above-mentioned particular detection temperature
The difference that signal out and the signal detected at a temperature of above-mentioned relatively low detection carry out Mathematical treatment to obtain.
27. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that in the case where not detecting signal at a temperature of the detection for being higher than above-mentioned particular detection temperature, it is contemplated that
Signal is not detected at a temperature of being higher than the detection of above-mentioned particular detection temperature, is detected according at a temperature of above-mentioned particular detection
Signal determine have above-mentioned particular detection temperature target nucleic acid sequence presence.
28. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that in the presence of the target nucleic acid sequence with the detection temperature for being higher than above-mentioned specific plus testing temperature, above-mentioned
In method using at least three target nucleic acid sequence in different detection temperature detection samples, there is above-mentioned particular detection in order to determine
The presence of the target nucleic acid sequence of temperature and utilize at least one a reference value.
29. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 28
Method, which is characterized in that in order to determine the presence of the target nucleic acid sequence with above-mentioned particular detection temperature, above-mentioned using different
At least one a reference value utilized in the method for at least three target nucleic acid sequence in said reference value in temperature detection sample is detected,
(i) it is trained together with corresponding signal-generation mechanism in other different reaction vessels of the reaction vessel in above-mentioned steps (a)
All combinations of the target nucleic acid sequence with the detection temperature higher than above-mentioned particular detection temperature are supported, (ii) is higher than above-mentioned specific
After detecting signal at a temperature of 1 or more the detection temperature and above-mentioned particular detection of detection temperature, (iii) is by finding out in height
It the signal that is detected at a temperature of 1 or more detection of above-mentioned particular detection temperature and is examined at a temperature of above-mentioned particular detection
The difference between signal measured obtains said reference value.
30. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that above-mentioned 1 reaction vessel further includes at least one attachment device, above-mentioned at least one attachment device difference
Including 3 additional signal-generation mechanisms for detecting the target nucleic acid sequence other than above-mentioned 3 target nucleic acid sequences, In
The above-mentioned signal generated by a device of at least three signal-generation mechanism is mutually distinguishable in said vesse, by different types of
Detector detects above-mentioned signal respectively.
31. utilizing at least three target nucleic acid sequence in different detection temperature detection samples according to embodiment 17
Method, which is characterized in that above-mentioned target nucleic acid sequence includes nucleotide diversity.
32. a kind of method using 2 target nucleic acid sequences in different detection temperature and melting analysis detection sample, feature
It is, comprising:
Step (a), 2 signal-generation mechanisms in 1 reaction vessel and for detecting above-mentioned 2 target nucleic acid sequences
Said sample is cultivated together, and detects generated signal using the detector of single type, by corresponding signal-generation machine
Structure detects each above-mentioned target nucleic acid sequence, and one of above-mentioned 2 target nucleic acid sequences has true by corresponding signal-generation mechanism
Fixed relatively high detection temperature, another has the relatively low detection determined by corresponding above-mentioned signal-generation mechanism
Temperature, above-mentioned relatively high detection temperature are that can generate for the target nucleic acid sequence with above-mentioned relatively high detection temperature
The temperature of signal, above-mentioned relatively low detection temperature are that can generate for the target nucleic acid with above-mentioned relatively low detection temperature
The temperature of the signal of the signal of sequence and the target nucleic acid sequence with above-mentioned relatively high detection temperature, by above-mentioned 2 signals-
The signal that generation mechanism generates is not distinguished by the detector of above-mentioned single type, is carried out at a temperature of above-mentioned relatively high detection
State detection；
Step (b), in order to determine the presence of the target nucleic acid sequence with above-mentioned relatively low detection temperature, in prescribed limit
At a temperature of, carry out the melting analysis to the cultivation results object of above-mentioned steps (a)；And
Step (c), being determined according to the signal detected in above-mentioned steps (a) has above-mentioned relatively high detection temperature
The presence of target nucleic acid sequence, in above-mentioned steps (b), being determined using melting analysis result has the target of relatively low detection temperature
The presence of nucleic acid sequence.
33. utilizing 2 target nucleic acids in different detection temperature and melting analysis detection sample according to embodiment 32
The method of sequence, which is characterized in that carry out above-mentioned steps (a) during with the amplification of signal of nucleic acid amplification.
34. utilizing 2 target nucleic acids in different detection temperature and melting analysis detection sample according to embodiment 32
The method of sequence, which is characterized in that carry out above-mentioned steps (a) during the amplification of signal of free nucleic acid amplification.
35. utilizing 2 target nucleic acids in different detection temperature and melting analysis detection sample according to embodiment 32
The method of sequence, which is characterized in that signal-generation mechanism for each above-mentioned target nucleic acid sequence is to depend on dimer shape
At mode generate signal-generation mechanism of signal.
36. utilizing 2 target nucleic acids in different detection temperature and melting analysis detection sample according to embodiment 32
The method of sequence, which is characterized in that for each above-mentioned target nucleic acid sequence signal-generation mechanism be utilize with dependent on it is upper
The mode for stating the cutting of the mediation oligonucleotides of target nucleic acid sequence specific hybrid forms signal-generation mechanism of dimer.
37. utilizing 2 target nucleic acids in different detection temperature and melting analysis detection sample according to embodiment 32
The method of sequence, which is characterized in that for signal-generation mechanism of the target nucleic acid sequence with above-mentioned relatively high detection temperature
For based on detection oligonucleotides cutting signal-generation mechanism, for it is above-mentioned it is relatively low detection temperature target nucleic acid
Signal-generation mechanism of sequence is the signal-generation mechanism formed based on dimer.
38. utilizing 2 target nucleic acids in different detection temperature and melting analysis detection sample according to embodiment 32
The method of sequence, which is characterized in that for signal-generation mechanism of the target nucleic acid sequence with above-mentioned relatively high detection temperature
For based on detection oligonucleotides cutting signal-generation mechanism, for it is above-mentioned it is relatively low detection temperature target nucleic acid
Signal-generation mechanism of sequence is to utilize to cut dependent on the mediation oligonucleotides of above-mentioned target nucleic acid sequence specific hybrid
The mode cut forms signal-generation mechanism of dimer.
39. utilizing 2 target nucleic acids in different detection temperature and melting analysis detection sample according to embodiment 32
The method of sequence, which is characterized in that above-mentioned 2 signal-generation mechanisms include identical label, from the signal of above-mentioned label not by
The detector of single type is distinguished.
40. utilizing 2 target nucleic acids in different detection temperature and melting analysis detection sample according to embodiment 32
The method of sequence, which is characterized in that above-mentioned target nucleic acid sequence includes nucleotide diversity.
41. a kind of method using at least three target nucleic acid sequence in detection temperature and melting analysis detection sample, feature
It is, comprising:
Step (a), at least three signal-in 1 reaction vessel and for detecting above-mentioned at least three target nucleic acid sequence
Generation mechanism cultivates said sample together, and detects produced signal using the detector of single type, by corresponding signal-
Generation mechanism detects above-mentioned at least three target nucleic acid sequence respectively, and above-mentioned at least three target nucleic acid sequence is respectively provided with by corresponding
The different detection temperature that above-mentioned signal-generation mechanism determines, above-mentioned detection temperature is that can not only generate with above-mentioned detection temperature
The signal of the target nucleic acid sequence of degree and can also generate for higher than above-mentioned detection temperature detection temperature target nucleic acid sequence
The temperature of the signal of column, the signal generated by above-mentioned signal-generation mechanism are not distinguished by the detector of above-mentioned single type, are passed through
Detection temperature analysis detects a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence, in above-mentioned at least three target nucleic acid
The detection temperature of above-mentioned a part of target nucleic acid sequence in sequence and 1 or more detection temperature higher than above-mentioned detection temperature
Carry out above-mentioned detection；
Step (b), in order to be determined other than above-mentioned a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence
Other target nucleic acid sequences presence, prescribed limit at a temperature of, unwinding point is carried out to the cultivation results objects of above-mentioned steps (a)
Analysis；And
Step (c) (i) is determined according to the signal detected in above-mentioned steps (a) above-mentioned in above-mentioned target nucleic acid sequence
The presence of a part of target nucleic acid sequence, when determining in above-mentioned a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence
Target nucleic acid sequence with particular detection temperature there are when, then according to being higher than 1 or more of above-mentioned particular detection temperature
Difference determination between the signal detected at a temperature of detection and the signal detected at a temperature of above-mentioned particular detection has
The presence of the target nucleic acid sequence of above-mentioned particular detection temperature, above-mentioned particular detection temperature is opposite highest in above-mentioned detection temperature
When detecting temperature, being determined according to the signal detected at a temperature of above-mentioned particular detection has the target nucleus of above-mentioned particular detection temperature
The presence of acid sequence, (ii) determine above-mentioned at least three target nucleic acid sequence in above-mentioned steps (b), according to the result of melting analysis
In other target nucleic acid sequences other than above-mentioned a part of target nucleic acid sequence presence.
42. utilizing at least three target nucleic acid in detection temperature and melting analysis detection sample according to embodiment 41
The method of sequence, which is characterized in that after determining the presence with the target nucleic acid sequence of opposite highest detection temperature first, by drop
Sequence arrangement successively determines that the existing mode with the target nucleic acid sequence of relatively low detection temperature carries out above-mentioned steps (c).
43. utilizing at least three target nucleic acid in detection temperature and melting analysis detection sample according to embodiment 41
The method of sequence, which is characterized in that carry out above-mentioned steps (a) during with the amplification of signal of nucleic acid amplification.
44. utilizing at least three target nucleic acid in detection temperature and melting analysis detection sample according to embodiment 41
The method of sequence, which is characterized in that carry out above-mentioned steps (a) during the amplification of signal of free nucleic acid amplification.
45. utilizing at least three target nucleic acid in detection temperature and melting analysis detection sample according to embodiment 41
The method of sequence, which is characterized in that signal-generation mechanism for each above-mentioned target nucleic acid sequence is to depend on dimer shape
At mode generate signal-generation mechanism of signal.
46. utilizing at least three target nucleic acid in detection temperature and melting analysis detection sample according to embodiment 41
The method of sequence, which is characterized in that for each above-mentioned target nucleic acid sequence signal-generation mechanism be utilize with dependent on it is upper
The mode for stating the cutting of the mediation oligonucleotides of target nucleic acid sequence specific hybrid forms signal-generation mechanism of dimer.
47. utilizing at least three target nucleic acid in detection temperature and melting analysis detection sample according to embodiment 41
The method of sequence, which is characterized in that for signal-generation machine of the target nucleic acid sequence with above-mentioned opposite highest temperature detection temperature
Structure is signal-generation mechanism of the cutting based on detection oligonucleotides, for signal-generation machine of remaining above-mentioned target nucleic acid sequence
Structure is the signal-generation mechanism formed based on dimer.
48. utilizing at least three target nucleic acid in detection temperature and melting analysis detection sample according to embodiment 41
The method of sequence, which is characterized in that for signal-generation machine of the target nucleic acid sequence with above-mentioned opposite highest temperature detection temperature
Structure is signal-generation mechanism of the cutting based on detection oligonucleotides, for signal-generation machine of remaining above-mentioned target nucleic acid sequence
Structure is to utilize to form dimerization in the way of depending on the cutting of the mediation oligonucleotides of above-mentioned target nucleic acid sequence specific hybrid
Signal-generation mechanism of object.
49. utilizing at least three target nucleic acid in detection temperature and melting analysis detection sample according to embodiment 41
The method of sequence, which is characterized in that above-mentioned target nucleic acid sequence includes nucleotide diversity.
50. a kind of kit using 2 target nucleic acid sequences in different detection temperature detection samples, which is characterized in that packet
It includes:
(a) 2 signal-generation mechanisms, for detecting above-mentioned 2 target nucleic acid sequences；And
(b) specification records method described in any one of embodiment 1 to 15.
51. a kind of kit using different detection temperature analysis sample nucleic acid sequence nucleotide polymorphisms genotype,
It is characterised by comprising:
(a) signal-generation mechanism, for detecting nucleotide polymorphisms allele；And
(b) specification records method described in embodiment 16.
52. a kind of utilize at least 3 nucleic acid sequence kits in different detection temperature detection samples characterized by comprising
(a) at least three signal-generation mechanism, for detecting above-mentioned at least three target nucleic acid sequence；And
(b) specification records method described in any one of embodiment 17 to 31.
53. a kind of kit using at least two target nucleic acid sequence in different detection temperature and melting analysis detection sample,
It is characterised by comprising:
(a) 2 signal-generation mechanisms, for detecting above-mentioned 2 target nucleic acid sequences；And
(b) specification, record embodiment 32 to 40 any one of described in method.
54. a kind of kit using at least three target nucleic acid sequence in detection temperature and melting analysis detection sample, special
Sign is, comprising:
(a) at least three signal-generation mechanism, for detecting above-mentioned 3 target nucleic acid sequences；And
(b) specification records method described in any one of embodiment 41 to 49.
55. a kind of computer readable storage medium, comprising the instruction for running processor, above-mentioned processor is for executing
The existing method of 2 target nucleic acid sequences in sample is determined using different detection temperature, above-mentioned computer readable storage medium
It is characterized in that, the above-mentioned existing method that 2 target nucleic acid sequences in sample are determined using different detection temperature includes:
Step (a) receives the signal detected at a temperature of relatively high detection and at a temperature of relatively low detection
The signal detected detects each above-mentioned target nucleic acid sequence, above-mentioned 2 target nucleic acid sequences by corresponding signal-generation mechanism
A sequence there is the relatively high detection temperature determined by corresponding signal-generation mechanism, another sequence, which has, borrows
The above-mentioned relatively low detection temperature for helping corresponding above-mentioned signal-generation mechanism to determine, above-mentioned relatively high detection temperature are energy
Enough temperature for generating the signal for the target nucleic acid sequence with above-mentioned relatively high detection temperature, above-mentioned relatively low detection temperature
Degree for can generate for it is above-mentioned it is relatively low detection temperature target nucleic acid sequence signal and with it is above-mentioned relatively
The temperature of the signal of the target nucleic acid sequence of the Supreme People's Procuratorate's testing temperature；And
Step (b), according to received above-mentioned signal determine the presence of above-mentioned 2 target nucleic acid sequences, (i) according to above-mentioned
The signal detected at a temperature of relatively high detection determines the presence with the target nucleic acid sequence of above-mentioned relatively high detection temperature,
(ii) according to the signal detected at a temperature of higher than above-mentioned relatively high detection and in above-mentioned relatively low detection temperature
Difference between the signal detected under degree determines the presence with the target nucleic acid sequence of above-mentioned relatively low detection temperature.
56. a kind of device using target nucleic acid sequence in different detection temperature detection samples characterized by comprising
(a) computer processor；And
(b) computer readable storage medium described in embodiment 55, couples with above-mentioned computer processor.
57. a kind of computer program is run for determining the processor of the method for 2 target nucleic acid sequences and storage in sample
In computer readable storage medium, above-mentioned computer program is characterized in that, the side of 2 target nucleic acid sequences in above-mentioned decision sample
Method includes:
Step (a) receives the signal detected at a temperature of relatively high detection and at a temperature of relatively low detection
The signal detected detects each above-mentioned target nucleic acid sequence by corresponding signal-generation mechanism, utilizes the detection of single type
Device detects generated above-mentioned signal, and one in above-mentioned 2 target nucleic acid sequences is with true by corresponding signal-generation mechanism
Fixed relatively high detection temperature, another is above-mentioned relatively low with determining by corresponding above-mentioned signal-generation mechanism
Temperature is detected, above-mentioned relatively high detection temperature is that can generate for the target nucleic acid sequence with above-mentioned relatively high detection temperature
The temperature of the signal of column, above-mentioned relatively low detection temperature are that can generate for the target with above-mentioned relatively low detection temperature
The temperature of the signal of the signal of nucleic acid sequence and the target nucleic acid sequence with above-mentioned relatively high detection temperature；And
Step (b), according to received above-mentioned signal determine the presence of above-mentioned 2 target nucleic acid sequences, (i) according to above-mentioned
The signal detected at a temperature of relatively high detection determines the presence with the target nucleic acid sequence of above-mentioned relatively high detection temperature,
(ii) according to the signal detected at a temperature of higher than above-mentioned relatively high detection and at a temperature of above-mentioned relatively low detection
Difference between the signal detected determines the presence with the target nucleic acid sequence of above-mentioned relatively low detection temperature.
58. a kind of computer readable storage medium, comprising the instruction for running processor, above-mentioned processor is for executing
The method that the nucleotide polymorphisms genotype of sample nucleic acid sequence is analyzed using different detection temperature, above-mentioned computer
Readable storage medium storing program for executing is characterized in that, it is above-mentioned using different detection temperature to the nucleotide polymorphisms gene of sample nucleic acid sequence
The method that type is analyzed includes:
Step (a) receives the signal detected at a temperature of relatively high detection and at a temperature of relatively low detection
The signal detected detects each above-mentioned nucleotide polymorphisms allele, above-mentioned nucleosides by corresponding signal-generation mechanism
A gene in sour polymorphic allele is above-mentioned relatively high with determining by corresponding above-mentioned signal-generation mechanism
Temperature is detected, another gene has the above-mentioned relatively low detection temperature determined by corresponding above-mentioned signal-generation mechanism,
Above-mentioned relatively high detection temperature is that can generate for the nucleotide polymorphisms gene with above-mentioned relatively high detection temperature
Signal temperature, it is above-mentioned it is relatively low detection temperature be can generate for it is above-mentioned it is relatively low detect temperature nucleosides
The temperature of the signal of the signal of sour Genetic polymorphism and the nucleotide polymorphisms gene with above-mentioned relatively high detection temperature；
And
Step (b) determines nucleotide polymorphisms genotype according to the difference between received signal.
59. a kind of computer readable storage medium, comprising the instruction for running processor, above-mentioned processor is for executing
The presence of at least three target nucleic acid sequence in sample, the spy of above-mentioned computer readable storage medium are determined using different detection temperature
Sign is, above-mentioned to include: using existing method of the different detection temperature at least three target nucleic acid sequence in determining sample
Step (a) receives the signal detected at a temperature of at least three detection, by corresponding signal-generation mechanism point
Above-mentioned at least three target nucleic acid sequence is not detected, and above-mentioned at least three target nucleic acid sequence is respectively provided with by corresponding above-mentioned signal-
The different detection temperature that generation mechanism determines, above-mentioned detection temperature is that can not only generate the target nucleic acid with above-mentioned detection temperature
The signal of sequence and signal for the target nucleic acid sequence with the detection temperature higher than above-mentioned detection temperature can also be generated
Temperature, the signal generated by above-mentioned signal-generation mechanism are not distinguished by the detector of above-mentioned single type, in each different detections
At a temperature of carry out above-mentioned detection；And
Step (b) determines the presence of above-mentioned at least three target nucleic acid sequence according to received signal, in above-mentioned at least three
Determined in target nucleic acid sequence the target nucleic acid sequence with particular detection temperature there are when, according to being higher than particular detection temperature
Difference between the signal detected at a temperature of 1 or more detection and the signal detected at a temperature of above-mentioned particular detection
Determine the presence with the target nucleic acid sequence of above-mentioned relatively low detection temperature, the above-mentioned specific inspection in above-mentioned detection temperature
When testing temperature is opposite highest detection temperature, above-mentioned target nucleic acid is determined according to the signal detected at a temperature of above-mentioned particular detection
The presence of sequence.
Preferred embodiment of the invention is described in detail above, can carry out deformation and modification according to the principles of the present invention, this
It is it will be apparent that the scope of the present invention can be according to appended hair for general technical staff of the technical field of the invention
Bright claimed range and its equivalent technical solutions define.
Sequence table
<110> SEEGENE, INC.
<120>it is detected using the target nucleic acid sequence of different detection temperature
<130> PP140137
<150> KR PCT/KR2014/006714
<151> 2014-07-23
<150> KR 10-2014-0037310
<151> 2014-03-28
<150> US 61/979,545
<151> 2014-04-15
<150> KR PCT/KR2014/004173
<151> 2014-05-09
<160> 18
<170> KopatentIn 2.0
<210> 1
<211> 37
<212> DNA
<213>artificial sequence
<220>
<223> NG-F
<220>
<221> misc_feature
<222> (22)..(26)
<223>n refers to deoxyinosine
<400> 1
tacgcctgct actttcacgc tnnnnngtaa tcagatg 37
<210> 2
<211> 37
<212> DNA
<213>artificial sequence
<220>
<223> NG-R
<220>
<221> misc_feature
<222> (22)..(26)
<223>n refers to deoxyinosine
<400> 2
caatggatcg gtatcactcg cnnnnncgag caagaac 37
<210> 3
<211> 34
<212> DNA
<213>artificial sequence
<220>
<223> NG-PTO
<400> 3
gtacgcgata cgggcccctc attggcgtgt ttcg 34
<210> 4
<211> 39
<212> DNA
<213>artificial sequence
<220>
<223> NG-CTO
<400> 4
tttttttttt tttttttttg tactgcccgt atcgcgtac 39
<210> 5
<211> 40
<212> DNA
<213>artificial sequence
<220>
<223> CT-F
<220>
<221> misc_feature
<222> (26)..(30)
<223>n refers to deoxyinosine
<400> 5
gagttttaaa atgggaaatt ctggtnnnnn tttgtataac 40
<210> 6
<211> 40
<212> DNA
<213>artificial sequence
<220>
<223> CT-R
<220>
<221> misc_feature
<222> (26)..(30)
<223>n refers to deoxyinosine
<400> 6
ccaattgtaa tagaagcatt ggttgnnnnn ttattggaga 40
<210> 7
<211> 39
<212> DNA
<213>artificial sequence
<220>
<223> CT-PTO
<400> 7
gattacgcga ccgcatcaga agctgtcatt ttggctgcg 39
<210> 8
<211> 39
<212> DNA
<213>artificial sequence
<220>
<223> CT-CTO
<400> 8
gcgctggata ccctggacga tatgtgcggt cgcgtaatc 39
<210> 9
<211> 26
<212> DNA
<213>artificial sequence
<220>
<223> CT-P
<400> 9
catcagaagc tgtcattttg gctgcg 26
<210> 10
<211> 32
<212> DNA
<213>artificial sequence
<220>
<223> M677-F
<220>
<221> misc_feature
<222> (18)..(22)
<223>n refers to deoxyinosine
<400> 10
ccaccccgaa gcagggannn nngaggctga cc 32
<210> 11
<211> 34
<212> DNA
<213>artificial sequence
<220>
<223> M677-R
<220>
<221> misc_feature
<222> (20)..(24)
<223>n refers to deoxyinosine
<400> 11
caagtgatgc ccatgtcggn nnnngccttc acaa 34
<210> 12
<211> 37
<212> DNA
<213>artificial sequence
<220>
<223> M677-W-PTO
<400> 12
ggtcccgacg ttagctcccg cagacacctt ctccttc 37
<210> 13
<211> 39
<212> DNA
<213>artificial sequence
<220>
<223> M677-W-CTO
<400> 13
cctcggtgcc acgccatcgg ttcttctaac gtcgggacc 39
<210> 14
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> M677-M-PTO
<400> 14
acgtcgattc gcactcccgc agacaccttc tccttcaa 38
<210> 15
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> M677-M-CTO
<400> 15
tttttttttt tttttttttt tattctgcga atcgacgt 38
<210> 16
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> MG-F
<220>
<221> misc_feature
<222> (23)..(27)
<223>n refers to deoxyinosine
<400> 16
aaaacccacg gaaatgatga gannnnnatt ggttctac 38
<210> 17
<211> 41
<212> DNA
<213>artificial sequence
<220>
<223> MG-R
<220>
<221> misc_feature
<222> (27)..(30)
<223>n refers to deoxyinosine
<400> 17
ctcgttaatt tacctattcc attttgnnnn nctgataaaa g 41
<210> 18
<211> 28
<212> DNA
<213>artificial sequence
<220>
<223> MG-P
<400> 18
gagttctttc aagaacagca agaggtgt 28
Claims (13)
1. a kind of method using 2 target nucleic acid sequences in different detection temperature and melting analysis detection sample, which is characterized in that
Include:
Step (a), in 1 reaction vessel together with 2 signal-generation mechanisms for detecting above-mentioned 2 target nucleic acid sequences
Said sample is cultivated, and detects generated signal using the detector of single type, is examined by corresponding signal-generation mechanism
Each above-mentioned target nucleic acid sequence is surveyed, one of above-mentioned 2 target nucleic acid sequences has determining by corresponding signal-generation mechanism
Relatively high detection temperature, another has the relatively low detection temperature determined by corresponding above-mentioned signal-generation mechanism,
Above-mentioned relatively high detection temperature is the signal that can be generated for the target nucleic acid sequence with above-mentioned relatively high detection temperature
Temperature, it is above-mentioned it is relatively low detection temperature be can generate for it is above-mentioned it is relatively low detect temperature target nucleic acid sequence
Signal and with it is above-mentioned it is relatively high detection temperature target nucleic acid sequence signal temperature, by above-mentioned 2 signal-generations
The signal that mechanism generates is not distinguished by the detector of above-mentioned single type, and above-mentioned inspection is carried out at a temperature of above-mentioned relatively high detection
It surveys；
Step (b), in order to determine the presence of the target nucleic acid sequence with above-mentioned relatively low detection temperature, in the temperature of prescribed limit
Under degree, the melting analysis to the cultivation results object of above-mentioned steps (a) is carried out；And
Step (c), being determined according to the signal detected in above-mentioned steps (a) has the target nucleus of above-mentioned relatively high detection temperature
The presence of acid sequence, in above-mentioned steps (b), being determined using melting analysis result has the target nucleic acid of relatively low detection temperature
The presence of sequence.
2. according to claim 1 utilize 2 target nucleic acid sequences in different detection temperature and melting analysis detection sample
Method, which is characterized in that signal-generation mechanism for each above-mentioned target nucleic acid sequence is the side to be formed dependent on dimer
Signal-generation mechanism of formula generation signal.
3. according to claim 1 utilize 2 target nucleic acid sequences in different detection temperature and melting analysis detection sample
Method, which is characterized in that for it is above-mentioned it is relatively high detection temperature target nucleic acid sequence signal-generation mechanism be based on
Signal-the generation mechanism for detecting the cutting of oligonucleotides, for the target nucleic acid sequence with above-mentioned relatively low detection temperature
Signal-generation mechanism is the signal-generation mechanism formed based on dimer.
4. according to claim 1 utilize 2 target nucleic acid sequences in different detection temperature and melting analysis detection sample
Method, which is characterized in that above-mentioned 2 signal-generation mechanisms include identical label, from the signal of above-mentioned label not by unitary class
The detector of type is distinguished.
5. a kind of method using at least three target nucleic acid sequence in detection temperature and melting analysis detection sample, which is characterized in that
Include:
Step (a), at least three signal-generation in 1 reaction vessel and for detecting above-mentioned at least three target nucleic acid sequence
Said sample is cultivated together by mechanism, and detects produced signal using the detector of single type, by corresponding signal-generation
Mechanism detects above-mentioned at least three target nucleic acid sequence respectively, and above-mentioned at least three target nucleic acid sequence is respectively provided with by corresponding above-mentioned
The different detection temperature that signal-generation mechanism determines, above-mentioned detection temperature is that can not only generate with above-mentioned detection temperature
It the signal of target nucleic acid sequence and can also generate for the target nucleic acid sequence with the detection temperature higher than above-mentioned detection temperature
The temperature of signal, the signal generated by above-mentioned signal-generation mechanism are not distinguished by the detector of above-mentioned single type, pass through detection
Temperature analysis detects a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence, in above-mentioned at least three target nucleic acid sequence
In above-mentioned a part of target nucleic acid sequence detection temperature and higher than above-mentioned detection temperature 1 or more detection temperature into
The above-mentioned detection of row；
Step (b), in order to determine its other than above-mentioned a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence
The presence of his target nucleic acid sequence, prescribed limit at a temperature of, melting analysis is carried out to the cultivation results objects of above-mentioned steps (a)；
And
Step (c) (i) determines above-mentioned one in above-mentioned target nucleic acid sequence according to the signal detected in above-mentioned steps (a)
The presence for dividing target nucleic acid sequence, when determination has in above-mentioned a part of target nucleic acid sequence in above-mentioned at least three target nucleic acid sequence
The target nucleic acid sequence of particular detection temperature there are when, then according to be higher than above-mentioned particular detection temperature 1 or more detection
At a temperature of difference between the signal detected and the signal detected at a temperature of above-mentioned particular detection determine have it is above-mentioned
The presence of the target nucleic acid sequence of particular detection temperature, above-mentioned particular detection temperature is opposite highest detection in above-mentioned detection temperature
When temperature, being determined according to the signal detected at a temperature of above-mentioned particular detection has the target nucleic acid sequence of above-mentioned particular detection temperature
The presence of column, (ii) are determined in above-mentioned at least three target nucleic acid sequence in above-mentioned steps (b) according to the result of melting analysis
The presence of other target nucleic acid sequences other than above-mentioned a part of target nucleic acid sequence.
6. according to claim 5 utilize at least three target nucleic acid sequence in detection temperature and melting analysis detection sample
Method, which is characterized in that after determining the presence with the target nucleic acid sequence of opposite highest detection temperature first, to arrange in descending order
Successively determine that the existing mode with the target nucleic acid sequence of relatively low detection temperature carries out above-mentioned steps (c).
7. according to claim 5 utilize at least three target nucleic acid sequence in detection temperature and melting analysis detection sample
Method, which is characterized in that signal-generation mechanism for each above-mentioned target nucleic acid sequence is the side to be formed dependent on dimer
Signal-generation mechanism of formula generation signal.
8. according to claim 5 utilize at least three target nucleic acid sequence in detection temperature and melting analysis detection sample
Method, which is characterized in that signal-generation mechanism for the target nucleic acid sequence with above-mentioned opposite highest temperature detection temperature is base
In signal-generation mechanism of the cutting of detection oligonucleotides, signal-generation mechanism for remaining above-mentioned target nucleic acid sequence is base
In signal-generation mechanism that dimer is formed.
9. a kind of kit using at least two target nucleic acid sequence in different detection temperature and melting analysis detection sample, special
Sign is, comprising:
(a) 2 signal-generation mechanisms, for detecting above-mentioned 2 target nucleic acid sequences；And
(b) specification records method described in any one of Claims 1-4.
10. a kind of kit using at least three target nucleic acid sequence in detection temperature and melting analysis detection sample, feature exist
In, comprising:
(a) at least three signal-generation mechanism, for detecting above-mentioned 3 target nucleic acid sequences；And
(b) specification records method described in any one of claim 5 to 8.
11. a kind of method using 2 target nucleic acid sequences in different detection temperature detection samples characterized by comprising
Step (a) is cultivated together with signal-generation mechanism for detecting above-mentioned 2 target nucleic acid sequences in 1 reaction vessel
Said sample, and generated signal is detected using the detector of single type, it is detected by corresponding signal-generation mechanism each
A above-mentioned target nucleic acid sequence, one in above-mentioned 2 target nucleic acid sequences has and determines by corresponding above-mentioned signal-generation mechanism
Relatively high detection temperature, another has the relatively low detection determined by corresponding above-mentioned signal-generation mechanism
Temperature, above-mentioned relatively high detection temperature are that can generate for the target nucleic acid sequence with above-mentioned relatively high detection temperature
The temperature of signal, above-mentioned relatively low detection temperature are that can generate for the target nucleic acid with above-mentioned relatively low detection temperature
The temperature of the signal of sequence and the signal for the target nucleic acid sequence with above-mentioned relatively high detection temperature, by above-mentioned 2
The signal that signal-generation mechanism generates is not distinguished by the detector of above-mentioned single type, above-mentioned relatively high detection temperature with
And above-mentioned detection is carried out at a temperature of above-mentioned relatively low detection；And
Step (b) determines the presence of 2 target nucleic acid sequences according to the signal detected in above-mentioned steps (a), and (i) basis exists
The signal detected at a temperature of above-mentioned relatively high detection determines the target nucleic acid sequence with above-mentioned relatively high detection temperature
In the presence of (ii) is examined according to the signal gone out in above-mentioned relatively high detection temperature detection and at a temperature of above-mentioned relatively low detection
Difference between the signal measured determines the presence with the target nucleic acid sequence of above-mentioned relatively low detection temperature.
12. a kind of method of the nucleotide polymorphisms genotype using different detection temperature analysis sample nucleic acid sequences, special
Sign is, comprising:
Step (a), in 1 reaction vessel together with signal-generation mechanism for detecting nucleotide polymorphisms allele
Sample of the culture comprising the nucleic acid sequence containing above-mentioned mononucleotide polymorphism site, and detected using the detector of single type
Generated signal detects each above-mentioned nucleotide polymorphisms allele, above-mentioned nucleosides by corresponding signal-generation mechanism
One in sour polymorphic allele has the relatively high detection temperature determined by corresponding signal-generation mechanism, separately
One has the relatively low detection temperature determined by corresponding signal-generation mechanism, and above-mentioned relatively high detection temperature is
The temperature of the signal for the nucleotide polymorphisms allele with above-mentioned relatively high detection temperature, above-mentioned phase can be generated
It is that can generate for the nucleotide polymorphisms allele with above-mentioned relatively low detection temperature to lower detection temperature
Signal and needle set have the temperature of the signal of the nucleotide polymorphisms allele of above-mentioned relatively high detection temperature, by above-mentioned letter
Number-signal that generates of generation mechanism do not distinguished by the detector of above-mentioned single type, in above-mentioned relatively high detection temperature and
Above-mentioned detection is carried out at a temperature of above-mentioned relatively low detection；And
Step (b), according to the signal detected at a temperature of above-mentioned relatively high detection in above-mentioned steps (a) and above-mentioned
The difference between signal detected at a temperature of relatively low detection determines nucleotide polymorphisms genotype.
13. a kind of method using at least three target nucleic acid sequence in different detection temperature detection samples characterized by comprising
Step (a), at least three signal-generation in 1 reaction vessel and for detecting above-mentioned at least three target nucleic acid sequence
Said sample is cultivated together by mechanism, and detects generated signal using the detector of single type, by corresponding signal-production
Life structure detects above-mentioned at least three target nucleic acid sequence respectively, and above-mentioned at least three target nucleic acid sequence is respectively provided with by corresponding
The different detection temperature that signal-generation mechanism determines are stated, above-mentioned detection temperature is that can not only generate with above-mentioned detection temperature
Target nucleic acid sequence and can also generate for higher than above-mentioned detection temperature detection temperature target nucleic acid sequence signal
Temperature, the signal generated by above-mentioned signal-generation mechanism is not distinguished by the detector of above-mentioned single type, in each different inspections
Above-mentioned detection is carried out under testing temperature；And
Step (b) determines the presence of at least three target nucleic acid sequence according to the signal detected in above-mentioned steps (a), works as determination
Target nucleic acid sequence with the particular detection temperature in above-mentioned at least three target nucleic acid sequence there are when, according to be higher than it is above-mentioned
It the signal detected and is detected at a temperature of above-mentioned particular detection at a temperature of 1 or more detection of particular detection temperature
Difference between signal determines the presence with the target nucleic acid sequence of above-mentioned particular detection temperature, above-mentioned in above-mentioned detection temperature
When particular detection temperature is opposite highest detection temperature, determined according to the signal detected at a temperature of above-mentioned particular detection above-mentioned
The presence of target nucleic acid sequence.
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| PCT/KR2014/006714 WO2015147382A1 (en) | 2014-03-28 | 2014-07-23 | Detection of target nucleic acid sequences using different detection temperatures |
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